Volume 45 Number 15 7 August 2016 Pages 4035–4438

Chem Soc Rev

Chemical Society Reviews
www.rsc.org/chemsocrev

ISSN 0306-0012

REVIEW ARTICLE
Amy M. Wen and Nicole F. Steinmetz
Design of virus-based nanomaterials for medicine, biotechnology, and
energy
 Chem Soc Rev
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REVIEW ARTICLE View Journal | View Issue

Design of virus-based nanomaterials for medicine,

biotechnology, and energy
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Cite this: Chem. Soc. Rev., 2016,

45, 4074
Amy M. Wena and Nicole F. Steinmetz*abcde

Received 6th April 2015
DOI: 10.1039/c5cs00287g
www.rsc.org/chemsocrev
This review provides an overview of recent developments in ‘‘chemical virology.’’ Viruses, as materials, provide
unique nanoscale scaffolds that have relevance in chemical biology and nanotechnology, with diverse areas
of applications. Some fundamental advantages of viruses, compared to synthetically programmed materials,
include the highly precise spatial arrangement of their subunits into a diverse array of shapes and sizes and
many available avenues for easy and reproducible modification. Here, we will first survey the broad
distribution of viruses and various methods for producing virus-based nanoparticles, as well as engineering
principles used to impart new functionalities. We will then examine the broad range of applications and
implications of virus-based materials, focusing on the medical, biotechnology, and energy sectors. We
anticipate that this field will continue to evolve and grow, with exciting new possibilities stemming from
advancements in the rational design of virus-based nanomaterials.

a
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA. E-mail: nicole.steinmetz@case.edu
b
Department of Radiology, Case Western Reserve University, Cleveland, OH 44106, USA
c
Department of Materials Science and Engineering, Case Western Reserve University, Cleveland, OH 44106, USA
d
Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, OH 44106, USA
e
Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA

Amy M. Wen received her BSE Nicole F. Steinmetz is an

from Duke University in 2010. Assistant Professor of Biomedical
She then pursued her PhD at Engineering at Case Western
Case Western Reserve University Reserve University School of
under the supervision of Prof. Medicine. Her research interests
Nicole F. Steinmetz in the are in applying synthetic virology
Department of Biomedical Engi- approaches toward next-generation
neering. Her current research biomedicines and materials. She
interests focus on using bioinspired trained at RWTH-Aachen University
approaches to develop design in Germany (Masters in Molecular
guidelines and strategies for Biotechnology) and received her
therapeutic intervention. PhD in Bionanotechnology from
Amy M. Wen Nicole F. Steinmetz John Innes Centre, UK. She then
moved to the Scripps Research
Institute for her postdoctoral research under a NIH K99/R00 award.
Dr Steinmetz was named a 2015 Young Innovator of Cellular and
Biomolecular Engineering (BMES), and she is a 2014 Crain’s Cleveland
Business 40 under 40 honoree and a 2011 Mt. Sinai Scholar. She has
won many prestigious awards, including a 2016 American Cancer
Society Research Scholar Award, a 2015 NSF CAREER Award, and a
2014 Susan G. Komen Career Catalyst Grant. Dr Steinmetz has
authored more than 100 peer-reviewed journal articles, reviews, book
chapters, and patent applications; she has authored and edited books
on virus-based nanotechnology.

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1. Introduction
Nanoscale engineering is revolutionizing diverse disciplines in
science and technology. The use of viral scaffolds in particular
has led to advancements of scientific knowledge in self-assembly
and the development of novel materials with wide-ranging
applications. Viruses have been studied for more than 100 years,
and more than 5000 types of viruses have been discovered and
described. They come in a variety of shapes and sizes, and from a
chemist’s point of view they harbor many natural features that are
uniquely relevant to nanotechnology and nanoscience. To date,
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it has not been feasible to synthetically create nanoparticles of
comparable reproducibility, beauty, and utility. In a collaborative
effort, research into ‘‘physical or chemical virology’’ is directed
toward unraveling the processes of self-assembly and genome
packaging, understanding and controlling self-assembly of virus-
based materials into higher-order hierarchical structures, engi-
neering and studying virus-based and virus-like materials for
applications in the health and energy sectors, and scaled-up
manufacturing of such materials for applications in clinics and
in devices. In this review, we provide a general synopsis of the
engineering of virus-based and virus-like materials and we will
discuss the manifold and diverse applications of such. We start by
introducing the use of viruses from a materials perspective and
consider the methods for producing and modifying these particles.
We then survey some recent developments in the expansion of
their applications, with discussion focused on the utilization of
virus-based materials for medicine (delivery systems and contrast
agents), biotechnology (nanoreactors and sensing devices), and
energy (battery electrodes and storage devices). Finally, we assess
the opportunities and challenges for clinical or commercial
application of virus-inspired materials.
2. Viruses in a materials world
Viruses usually bring to mind devastating disease and bear a
negative connotation,1–3 especially with the recent outbreak of
Ebola in 2014 that spread quite rapidly and proved difficult to
control,4 as well as the current Zika virus outbreak that poses
issues with microcephaly in newborns and may also possibly
be linked to an increased risk of Guillain–Barré syndrome.5
Throughout history, infectious disease has plagued us, with the
earliest recordings found from over 3000 years ago of smallpox
in Egypt, India, and China.6 In fact, the mummy of Pharaoh
Ramses V, who died around 1157 BC, possesses pustules and
scarring reminiscent of smallpox infection. However, viruses
also have positive qualities, and there have been many advances
made in recent years in which nonpathogenic viruses and
engineered virus-based nanomaterials have been utilized as
three-dimensional scaffold materials for diagnostic and drug
delivery systems as well as technological devices. Viruses were
discovered to exist in 1892, and the first virus studied was the
plant virus tobacco mosaic virus (TMV).7 It was not long after
the discovery of viruses that they were considered for use in
biotechnology and medicine. Early in the twentieth century,
Frederick Twort and Felix d’Herelle independently reported the
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presence of bacterial viruses, or bacteriophages, and the idea of
phage therapy to treat bacterial infections quickly took shape in
the 1920’s, although it was mainly practiced in the Soviet
Union.8 The development of antibiotics largely overshadowed
phage therapy, but there may be a comeback due to the
increasing prevalence of antibiotics resistance,9 with benefits
of phage therapy including greater specificity, lower toxicities,
ability to disrupt bacterial biofilms, and ability to evolve to
combat resistance.10
Aside from phage therapy, there are many other avenues for
the use of viruses, and vaccines and gene therapy are likely the
first applications that come to mind. However, the potential
applications and current developments reach much farther.
Around 2000, a group of researchers that included chemists,
structural biologists, and virologists gave birth to a new field in
which viruses are used for nanotechnology by demonstrating
the ability to encapsulate materials within the capsid, address
them chemically, and order them into crystal structures.11–14
In this manner, viruses can simply be used as well-ordered
materials, separate from their normal role in infection. Most
viruses are made up of coat protein subunits that naturally
self-assemble into truly monodisperse particles. With more
understanding of the coat protein building blocks and chemical
biology, ever increasing complex assemblies can be programmed,
including nanoboomerang- and tetrapod-shaped virus materials.15
Large-scale production of viruses can be easily achieved through
propagation in their natural hosts or expression in a heterologous
system (see Section 2.2). Additionally, these particles come in a
variety of shapes and sizes16,17 that can function as nanoscaffolds
and can be easily and reproducibly modified.18 As shown in Fig. 1,
the most common architectures are icosahedrons, filaments, and
phage head-and-tail structures, but more diverse structures such as
spindle-, zipper-, and bottle-shaped viruses also exist.17,19
While there is the biotechnology arm where we seek to
engineer particles for applications in medicine and energy,
there is also a basic arm that investigates virus assembly and
structure. These two arms of research are interconnected,
with crosstalk between the two fields providing insights for
advancement. For example, study of the physics of the packing
signals of RNA viruses led to its application in the encapsulation
of therapeutics for nanomedical applications (see Sections 4.1.4
and 4.1.5).14,20 Additionally, fundamental understanding of the
interactions involved in particle self-assembly informed the
fabrication of novel imaging agents (see Section 4.1.2).21,22
Through multidisciplinary collaboration, the use of viral scaffolds
as unique materials for diverse applications can be realized.
2.1 Classification of viruses
To differentiate between viruses containing their native nucleic
acid, which are referred to as viral nanoparticles (VNPs), viruses
devoid of their nucleic acid are considered virus-like particles
(VLPs). Further classification of viruses can be based on a
number of features, including the shape and structure of their
capsids (as shown in Fig. 1), the type of nucleic acid they
contain (double-stranded (ds) or single-stranded (ss), RNA
or DNA), and their host species. Classical virology taxonomy
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Fig. 1 Some common viral architectures. Viruses come in diverse shapes and sizes, with icosahedral and helical symmetries as well as more complex
head-to-tail assemblies. For icosahedral viruses, examples of different triangulation numbers are shown (explained in Section 2.1), giving rise to different
capsid sizes and structures. An example of a virus for each architecture is given in italics below the figures.

utilizes the Baltimore classification of viruses, in which the
viruses are grouped both according to their genomes as well
as their method of replication.23 Fig. 2 illustrates the seven
different classifications of viruses, demonstrating how they
have evolved many different strategies for replication. However,
for the most part, we will be considering plant viruses and
bacteriophages (noninfectious particles) for use as materials,
making the native cargo of the capsids less relevant. Mammalian
viruses, such as adenovirus (class I – dsDNA virus) and adeno-
associated virus (AAV, class II – ssDNA virus), do offer many
advantages for applications in gene therapy, in which they can
be administered to make modifications to the genetic sequence
for therapeutic or prophylactic purposes (see Section 4.1.4).24
They also present opportunities in cancer immunotherapy, as
seen in the recent approval of T-VEC for treatment of melanoma
(see Section 4.1.3). Nevertheless, bacteriophage- and plant
virus-derived materials may offer advantages, as their manu-
facture is scalable through fermentation and molecular farming.
Additionally, these materials are not infectious toward mammals,
adding another layer of safety. Both these factors are important
considerations as we move toward clinical applications and
commercialization.
Other than genomic content and host species, shape and
size are important characteristics that should be considered for
the choice of material used. Section 4.1.1 highlights some of
the design guidelines for determining the desired properties for
delivery vectors for nanomedical applications. For icosahedral
viruses, the triangulation number, or T number, is one method
of classification that gives an indication as to their size (see
Fig. 1). The T number was first described by Donald Caspar and
Aaron Klug in 1962.25 By multiplying by 60, it can be used
to determine the number of coat proteins in a capsid. For
example, a T = 1 virus has 60 coat proteins, while a T = 4 virus
has 240 coat proteins. The proteins are clustered into pentamers
and hexamers, and a virus with icosahedral symmetry therefore
consists of 12 pentamers and 10(T  1) hexamers. Size plays a
factor in the transport and clearance behavior of a particle, as
well as the amount of cargo that can be carried and delivered to
a cell, a challenge with the smaller AAV (T = 1, B20 nm in
diameter). Additionally, the shape of the particles affects the
possible modifications and functions that could be applied to
the capsid. For example, icosahedrons have the advantage of
possessing an interior cavity that can be used for the infusion
and encapsulation of various payloads. On the other hand, high
aspect ratio particles can be used to form wires, which can then
be applied for energy applications. Overall, it is clear that there is
a diverse library of virus particles to select from, no matter the
application.

Fig. 2 Baltimore classification of viruses. With the Baltimore classification, viruses are classified based on their genomic material as well as their method
of replication.

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2.2 Production of viruses: fermentation, farming,
and cell culture
A variety of methods have been developed for the production
of virus particles, and we will discuss their manufacture in
bacteria, yeast, insect cells, plants, and using cell-free systems,
starting with one of the most widely utilized systems for the
rapid production of proteins with ease of scale-up, Escherichia
coli.26 Although other prokaryotic systems can also be considered,
such as Pseudomonas fluorescens,27 the wealth of knowledge
surrounding E. coli production makes it popular, and therefore
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it has also been widely applied for the production of VLPs. Viral
coat proteins can be expressed and spontaneously self-assembled
in the bacterial cells, and this has been demonstrated for bacterio-
phages, such as Qb28 and MS2,29 as well as for heterologous
expression of other viruses, such as plant virus-based TMV30 and
mammalian virus-based hepatitis B virus (HBV) core particles.31
It should be noted that while VLPs formed in this way do not
contain their own genomic content, they are also not ‘‘empty’’,
as they tend to package host nucleic acids. For example, about
25% of the mass of Qb VLPs (a system that has undergone
clinical testing) consists of E. coli RNA.32 For applications
where the packaged nucleic acid is undesirable, several methods
have been developed to remove the nucleic acid components
after particle assembly.33–35 These methods include treatment
with heavy metals such as lead acetate,33 incubation in alkaline
conditions for RNA hydrolysis,34 and induction of osmotic shock
using a high molarity sodium sulfate solution.35
Another approach for production of empty VLPs is in vitro
assembly of coat protein subunits after production in E. coli
and purification. In vitro assembly is also a way to overcome
challenges with insolubility of some eukaryotic capsid proteins
in the bacteria cells that result in their accumulation in inclusion
bodies.36 Digressing briefly, it is of significance to note that some
headway has been made with producing soluble eukaryotic coat
proteins with high yields and purity. This was demonstrated
recently with the plant virus cowpea chlorotic mosaic virus
(CCMV) through modulating several factors: an E. coli strain
resistant to chloramphenicol was utilized, which helps inhibit
protein transition to an insoluble state, and to give time for coat
proteins to fold and maintain solubility, lower temperature, lower
concentration of isopropyl-b-D-1-thiogalactopyranoside (IPTG) for
induction of expression, and E. coli with a slower rate of protein
synthesis were used.37 Returning to in vitro assembly, this
method has been demonstrated for a wide range of VLPs,
including those based on bacteriophages P2238 and PP7,39 plant
viruses potato virus X (PVX)40 and CCMV,37 and mammalian
viruses human papilloma virus (HPV)41 and human immuno-
deficiency virus (HIV).42 Some particles require a nucleic acid
template in order to self-assemble,30,39,40 but others can be
assembled to form empty capsids simply by altering conditions
such as temperature, pH, and molarity.37,38,41,42 For templated
self-assembly, TMV for example has an origin of assembly that
is thought to be required to drive its assembly,30 but other
filamentous particles may not have such sequence specificity.40,43
Knowledge of the self-assembly process of viruses can be
important for determining the types of payloads that can be
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encapsulated as well as informing the design of novel archi-
tectures.15 Assembly of pure, empty particles in vivo in high
yields is unique and has so far only been accomplished for
the plant virus cowpea mosaic virus (CPMV), which will be
discussed later with plant-based production systems.
In general, eukaryotic expression systems such as yeast, insect
cells, and plants may be favored for production of assembled
eukaryotic viruses as they are better able to secrete soluble
eukaryotic proteins and can perform post-translational modi-
fications, such as glycosylation, disulfide bond formation,
and proteolytic processing.44–46 Yeast expression systems work
similarly to bacterial systems and can also be scaled up using
fermentation technology. Some common yeast species that
have been developed for VLP production include Saccharomyces
cerevisiae and Pichia pastoris,47 and VLPs that have successfully
been produced in yeast include Qb,47 CCMV,48 and HPV,49
which incidentally is how Merck produces the vaccine Gardasil.
In addition to bacteria and yeast cells, cultures of insect
cells can also be utilized for VLP production. Baculovirus-based
expression systems can be cultured in insect cell lines such as
Spodoptera frugiperda (Sf) lines 9 and 21 and Trichoplusia ni
moth cells.50 These viruses contain a large genome that is
useful for incorporation of multiple genes of interest, but,
due to the lack of unique restriction sites, also requires alter-
native strategies such as combining the use of recombination
with shuttle vectors. This process tends to be a more time
consuming and lower yielding method. The baculovirus-based
expression system has been applied for the production of insect
viruses such as flock house virus (FHV), plant viruses including
CPMV, and mammalian viruses such as canine parvovirus
(CPV) and HPV,49,51–53 and it is GlaxoSmithKline’s method of
choice for producing its HPV vaccine Cervarix.
For production of plant VNPs and VLPs, plant-based expression
systems are frequently used. Some common plant virus-based
particles include red clover necrotic mottle virus (RCNMV),
BMV, CCMV, CPMV, PVX, and TMV. To produce VNPs, plants
can be infected by mechanical inoculation through applying
purified virus solutions, infected leaf samples, cDNA of the
virus genome, or even in vitro RNA transcripts to the leaves of
the plant after gentle abrasion.54,55 Agroinfiltration by injecting
a suspension of Agrobacterium tumefaciens bacteria into leaves
is also used for molecular farming in plants.56 These bacteria
transfer part of their tumor inducing plasmid into the plant
cell, which can be exploited for transient expression of genes of
interest. Of note, Medicago Inc. uses this plant-based approach
for the efficient production of VLP-based vaccines for influenza
and rabies, among others. Other therapeutics such as the
ZMAPP monoclonal antibody cocktail against Ebola virus (EBOV)
from Mapp Biopharmaceutical also utilize plant production with
agroinfiltration.57 Using such methods, replication of intact VNPs
such as BMV has been demonstrated.58 Additionally, viral capsids
of CPMV completely devoid of RNA (either virus or host) can be
produced in this way.59 Whereas BMV production utilized plasmids
that transiently express BMV RNAs to systemically infect plants, for
empty CPMV (eCPMV) VLP production, using plasmids encoding
just two proteins was found to be sufficient: VP60, which is a
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precursor to CPMV’s two coat proteins, and 24K proteinase
for proteolytic processing of VP60. As mentioned previously,
consistent empty VLP production in vivo has only been demon-
strated for the eCPMV platform. Aside from farming in the
plants themselves, some new technology that may be applied in
the future for the production of VLPs is the use of plant cell
packs for transient expression, where plant suspension cells are
packed into a ‘‘cookie’’ through suctioning, then Agrobacterium
containing the gene for the protein of interest applied.60 This
approach has been proven to produce high yields of recombi-
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3. Engineering virus-based scaffolds
Since viruses have evolved to protect and efficiently deliver their
nucleic acid cargo, they are able to withstand conditions
required for chemical modification and retain a long shelf life.
For modification, the interior cavity and exterior surface of
the viral capsids can both be utilized, allowing for the encap-
sulation of sensitive compounds and the display of targeting
moieties in precisely defined arrays, among other functions.
The beauty and utility of these particles have been recognized,
and efforts have been made toward mimicking these nanoscale

nant proteins and can be applied in a high throughput manner,

making it an attractive option for VLP expression.
Finally, as an alternative to the above in vivo approaches,
there has been some work involving VLP production using cell-
free systems where cellular machinery for transcription and
translation are used for protein expression in vitro. Some early
work in this area utilized a eukaryotic system based on rabbit
reticulocyte to study capsid assembly of hepatitis C virus (HCV),
HBV, and three primate lentiviruses, but the yields were quite
low (B10 ng mL1).61,62 Since then, exploration with an E. coli-
based system has achieved yields of around 400 ng mL1 for
MS2 and truncated HBV core antigen VLP production, with
almost complete solubility, making it an excellent platform for
rapid VLP production.63 Additionally, Qb VLPs were able to be
formed using this system through coproduction of its coat
protein with a cytotoxic A2 protein that is normally naturally
incorporated on the exterior of the capsid to facilitate infection,
demonstrating the advantage of a cell-free system for cytotoxic
protein production and regulating the relative expression of
multiple proteins.64 As the cost of cell-free systems goes down,
they may become more commonly applied for the production
of VLPs.
architectures through self-assembly of protein nanomaterials.65
The unique genetically encoded protein shell architecture of
virus-based scaffolds allows for a large range of techniques
that can be used to tailor and modify these materials. Among
the most frequently used of these that we will discuss are
genetic engineering, bioconjugation, infusion, biomineralization,
and self-assembly (Fig. 3). As engineering capabilities improve,
even greater diversities of virus-based and virus-like particles
can be created, expanding the possible applications of these
materials.
3.1 Genetic engineering
The coat proteins of VNPs are determined by their genetic
code. Nucleic acid sequences of viruses are relatively small,
and therefore many of their genomes have been sequenced
and are well characterized. Using genetic engineering, insertion
or replacement of residues can be performed to add functional
groups, with cysteine mutants being the most popular due
to possible disulfide linkages, association with gold, and
bioconjugation with thiol-selective chemistries.66–70 Insertion
of unnatural amino acids is also possible, allowing for more

Fig. 3 Techniques for modification of virus-based scaffolds. Simplified illustrations show common methods for internal and external modification of
viruses. To alter the composition of the protein capsid itself, genetic engineering can be used. With available exposed residues, bioconjugate chemistries
can be performed. Through pores in the structure, small cargo can be infused into the capsid and then retained by electrostatic interactions or by
reducing the pore size. Interactions of metal precursors with the capsid can be used to selectively direct mineralization on the interior or exterior surface.
Additionally, by taking advantage of self-assembly of the viral scaffold, cargo introduced during assembly can be encapsulated.

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diverse chemical modifications.71,72 Additionally, removal of
residues can be accomplished such that only a single unique
reactive site remains on the coat protein.73 Aside from single
residue modifications, larger changes such as insertion of
purification tags can also be accomplished. For example, due
to their affinity and coordination with nickel–nitrilotriacetic
acid (Ni–NTA), polyhistidine tags have been expressed on viral
capsids to serve as anchors for applications that include
tethering them to surfaces, attaching other particles such
as nanogold and iron oxide, and assembling higher-order
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structures.74–77 Display of other short peptide sequences have
been demonstrated, including epitopes for vaccines78–80 and
moieties for targeting receptors.81–83 Whole protein and
protein domain insertions can also be achieved,84,85 and even
virus hybrids consisting of coat proteins expressing different
proteins have been established through co-infection of plants
and verified by bimolecular fluorescence complementation.86
Aside from genetic engineering of the viral coat proteins,
tags such as the antibody binding peptide Z33 can be gene-
tically fused to fluorescent proteins, enzymes, and other
proteins of interest.87 In this particular example, assembly
of particles displaying the proteins can then be achieved by
means of an intermediary antibody specific to the viral coat
protein.
Fig. 4 Bioconjugation reactions that can be used for virus modification. Presented in the figure are some of the more common reactions for
functionalization of viruses. Other methods discussed in the text include atom-transfer radical polymerization, ring-opening metathesis polymerization,
and supramolecular interactions.
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3.2 Bioconjugate chemistry
Conjugation strategies targeting both natural and unnatural
amino acids on virus capsids allow for many possible modi-
fications that may not be achievable through genetic engi-
neering alone (Fig. 4). Both the interior and exterior surfaces
of many viruses have been shown to be amenable to chemical
modifications.88–90 Some common groups that can be function-
alized include lysine, aspartic/glutamic acid, cysteine, and tyrosine
residues, which lend themselves to standard bioconjugation
reactions involving N-hydroxysuccinimide (NHS) ester conjugation,
carbodiimide activation, Michael addition, and azo coupling
chemistries, respectively. Some alternatives to these natural
amino acids include replacing methionine residues with homo-
propargyl glycine (HPG) or azidohomoalanine (AHA) residues to
add alkyne or azide functionalities, respectively.71 Another
interesting method utilizes mutant tRNA synthetases to attach
unnatural amino acids to amber suppressor tRNAs for incor-
poration of these amino acids at amber stop codon sites.72,91
Among the amino acids incorporated in this way are O-methyl-
tyrosine, p-azidophenylalanine, p-acetylphenylalanine, p-benzoyl-
phenylalanine, 3-(2-naphthyl)alanine, and p-aminophenylalanine
(pAF). p-Azidophenylalanine and pAF are particularly noteworthy
due to providing azide and amine groups, respectively, for selective
coupling reactions. As can be noted, incorporation of azide and
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alkyne groups is an especially widespread strategy. These con-
jugation handles allow for copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC), an efficient and biocompatible procedure
that has found great versatility.92–96 Reaction without copper
catalysis can also be achieved by utilizing cyclooctyne derivatives,
which lower the activation barrier due to the ring strain.97
Additional reaction handles that have been utilized include
aldehydes and ketones for hydrazone or oxime condensation
reactions.88,98–100 Selective formation of aldehydes or ketones is
possible, where pyridoxal 5 0 -phosphate (PLP)-mediated trans-
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amination specific for the N-terminus leads to in situ oxidation
of the N-terminal amine.101 The formed ketone or an aldehyde
group can then be used to form stable oxime linkages with alkoxy-
amines. With the availability of a wide range of chemically
modifiable natural and unnatural amino acids, multiple func-
tional groups can be simultaneously incorporated within a single
virus-based particle to result in the formation of a versatile,
multifunctional platform.
One area of particle modification that is of great interest is
the formation of protein/polymer hybrid conjugates. Polyethylene
glycol (PEG) is a polymer frequently used for shielding biological
interactions, and attachment of PEG through aforementioned
bioconjugation techniques is fairly standard.89,102,103 More
sophisticated polymerization chemistry techniques have also
been shown to be applicable for polymer attachment to virus-
based particles, and more widespread application of these
could confer advantages of better efficiency and more control
over the polydispersity. Atom-transfer radical polymerization
(ATRP) is one such method, where small initiators can be
added to the particles first then polymerization from the capsid
carried out through the introduction of monomers, resulting in
easier purification of the smaller reagents as well as overcoming
challenges with steric hindrance of large bulky polymers.33,104,105
Incorporation of polymers using this method has proven to be
useful for the attachment or complexation of large payloads of
MR contrast agents, chemotherapeutics, and siRNA and for both
interior33,104 and exterior105 modification. Polymers could also
be synthesized first with ATRP before attachment to the viral
capsid, such as for the display of glycoproteins.106 Ring-
opening metathesis polymerization (ROMP) is another method
for biocompatible polymer synthesis, and it was utilized to
prepare water-soluble polynorbornene-based polymers with
strict size and architecture control, which had a good safety
profile when attached to Qb and delivered to fibroblast cells.107
Aside from chemistries that rely on covalent bonds, supra-
molecular interaction strategies can also be utilized for virus
modification. For example, by taking advantage of the hydro-
phobic interior of b-cyclodextrin that allows it to accommodate
a range of guest molecules, virus particles first modified with
b-cyclodextrin moieties can then be functionalized with derivatives
of such guest molecules. This approach has been demonstrated
using derivatives of adamantine for the display of an imaging
agent, chemotherapeutic drug, targeting ligand, and PEG
polymer.108 In a similar manner, charge-transfer interactions
between electron donors and acceptors can also be used for
derivatization of viral scaffolds. As an illustration, attachment
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of pyrene molecules allowed for particle functionalization
through interaction with electron-deficient dinitrophenyl and
pyridinium motifs.109
3.3 Infusion
The interior of viral capsids can be used as a cage for encap-
sulation of foreign cargo. Viruses are generally flexible and
contain pores that allow for diffusion of small molecules, such
as drugs and contrast agents, into and out of the capsid.
Retention of the molecules inside the capsid can then be
achieved through electrostatic and/or affinity interactions
with the nucleic acid within the shell93,110,111 or interactions
with polymers conjugated internally.33,104 Encapsulation of
molecules can also be accomplished by gating using pH or
metal ion concentration to trigger structural transitions. Using
the gating process, molecules are allowed to diffuse into the
particle under an environment where the capsid is in a swollen,
open conformation, and then the molecules are trapped within
the capsid as the pores are closed off through change in buffer
conditions.112–114 Depending on the desired application, the
molecules of interest can either remain encapsulated within
the particles or released over time. Examples of infusion for
imaging and drug delivery are described in Sections 4.1.2 and
4.1.5. Another application of infusion is for introducing metal
precursors into the capsid for interior mineralization, which is
discussed next.
3.4 Mineralization
Viral particles can also serve as templates in the biominerali-
zation process with unique size and shape control. Through
tuning electrostatics or the use of mineralization-directing
peptides, nucleation of precursor metal ions and subsequent
shape-constrained mineralization can be realized. Peptide
nucleators and binders were identified by screening using
phage display techniques against various substrates, such as
GaAs and ZnS,115,116 and shown to be highly specific. Minerali-
zation has been demonstrated for the interior11,117–119 and the
exterior120–122 surfaces of particles, as well as for both icosa-
hedral11,117,118,122 and rod-shaped119–121 viruses. These resulting
hybrid inorganic–organic materials find use in a variety of
functions, ranging from applications in energy as semiconductors
(see Section 4.3) to medicine as contrast agents (see Section 4.1).
3.5 Self-assembly
While we have mostly considered these virus-based particles as
intact scaffolds we can build from, they can also be taken apart
and reassembled, either with their natural genome or with
foreign cargo. There is a great breadth in the types of cargo that
can be encapsulated through self-assembly, including gold
nanoparticles, quantum dots, and photosensitizer drugs.21,123–126
Since coat proteins naturally self-assemble around negatively
charged nucleic acids, in general a more negative surface
charge results in more efficient encapsulation of the foreign
cargo.123 Native packaging mechanisms can also be utilized,
which was demonstrated with adding oligonucleotides mimicking
the origin of assembly for RCNMV’s packaging of its RNA on
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various nanoparticles to induce particle formation around the
different cargo.127 Size plays a factor in assembly due to its effect
on the radius of curvature, and differently sized cargo could result
in alterations in the morphology and physical characteristics of
the capsid.123–125
As seen in Section 3.1, coat proteins can be genetically
modified for incorporation of foreign protein cargo during
particle assembly. One study fused the coat proteins of CCMV
to elastin-like polypeptides (ELPs), which exhibit lower critical
solution temperature (LCST) behavior, and the investigators
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found two different self-assembly pathways from the resultant
hybrid.128 While VLPs consisting of 90 coat protein dimers were
formed under normal pH-induced self-assembly conditions,
when the NaCl concentration was increased to lower the ELP
transition temperature, the ELP-induced assembly resulted in
the formation of smaller particles consisting of 30 coat protein
dimers. Enzyme facilitation is another method for coat protein
modification, which was demonstrated through the use of
sortase A to covalently attach a protein with a C-terminal LPETG
tag to glycines at the N-terminus of CCMV coat proteins before
assembly for protein encapsulation.129 Specific binding inter-
actions with the coat protein can also be exploited for self-
assembly. Engineering of coiled-coil protein interactions was
established by introducing a lysine coil at the N-terminus of
CCMV that can associate with a glutamic acid coil at the
C-terminus of a fluorescent protein, which resulted in encap-
sulation of the protein when the two modified proteins were
combined.130 Introducing histidine tags that have affinity for
Ni–NTA is another method.77 Additionally, some interactions
that naturally exist for some particles include the association of
scaffold proteins with bacteriophage P22 that aids in viral
assembly131,132 and the binding of translational repression
operator proteins to RNA stem-loops within MS2 bacterio-
phages.14 Fusions to these proteins can then be utilized for
encapsulation of materials of interest, such as enzyme cascades
and therapeutic molecules.
4. Applications of virus-based particles
4.1 Medicine
Viruses have been applied broadly in medicine for diagnostic
and therapeutic purposes, and many are in the pipeline under-
going clinical trials for oncotherapy and as gene therapy
vectors.133,134 In fact, there is currently much excitement over
the recent approval of the oncotherapy talimogene laherparepvec
(T-VEC) manufactured by Amgen,135 and T-VEC will be discussed
in more detail in Section 4.1.3. Bacteriophages and plant
viruses are particularly attractive tools for biomedical applica-
tions because they do not replicate within mammalian cells,
and therefore the platforms may add another layer of safety. In
this section, we will explore the use of virus-based particles as
delivery vehicles targeted toward imaging and treatment of
diseases and as scaffolds that interact with the local environment,
which can be utilized for vaccines, immunotherapy, and tissue
engineering.
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4.1.1 Nanomedical viral engineering design rules. Some
important considerations for the design of viruses for applica-
tions in vivo include charge, shape, and surface ligand presen-
tation (Fig. 5). These design parameters affect their circulation
in the body as well as their cellular interactions and tissue
specificity. Some general principles have been established
specifically for virus-based particles,136 and we will highlight
some of the lessons here. It is important to note that although
these principles provide a good guideline, in vivo studies are
crucial for ascertaining how new proposed particles will behave
due to the intricacy and complexity of biological interactions,
which cannot be fully predicted through in vitro testing or
modeling.
In terms of charge, there appears to be a trend where virus-
based particles with negative surface charge tend to have shorter
circulation times. This was observed with negatively charged
CCMV, CPMV, and TMV, which have circulation half-lives of less
than 10 minutes.137–139 In comparison, the half-lives for positively
charged Qb and M13 are on the order of 4–5 hours.140,141 The
effect of charge on plasma clearance was made more evident
when much quicker clearance for both bacteriophages was observed
with the neutralization of their positive lysine residues.140,141
Additionally, the reverse study with a single amino acid substitu-
tion of glutamic acid residues with lysines using bacteriophage
l resulted in over a 1000-fold higher circulation time.142
A notable exception to this trend is PVX, which is expected to
be longer circulating based on its positive charge and abundance
of surface lysines but in fact has a quick clearance half-life of
around 10 minutes.143 A more recent study reported a negative
zeta potential for PVX,144 likely due to different buffer conditions
used, which indicates further investigation into the charge of PVX
Fig. 5 Design parameters to consider for nanoparticle engineering. Para-
meters include charge (positive or negative), shape and size (different
aspect ratio filaments and diameter spheres), shielding (self proteins/
peptides and polymers of various sizes and densities), and targeting
(ligands for receptors or environmental factors displayed on different
linkers and at various densities).
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under in vivo conditions is crucial for confirming whether or not
it defies convention.
Additional influences based on charge include altering how
particles interact with mammalian cells and tumor transport
rates. Due to the abundant presence of proteoglycan in the cell
membrane conferring a negative charge and collagen within
the tumor interstitial space conferring a positive charge,
positively charged particles are more likely to have enhanced
binding to mammalian cells145 and are better able to avoid
aggregation and penetrate tumor tissue.143,146 Some examples
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demonstrating these charge-based effects include polyarginine-
decorated CPMV found to be taken up eight times more
efficiently than native CPMV in a human cervical cancer cell
line145 and positively charged PVX shown to be able to penetrate
to the tumor core unlike negatively charged CPMV.143 In the
latter case, PVX’s filamentous nature also allowed it to better
avoid the macrophagocytic system, leading to greater tumor
homing.
It is likely that the shape and flexibility of PVX plays an
additional role in its ability to diffuse throughout the tumor.
A comparison between the diffusion profiles of a spherical and
rod-shaped particle was performed with CPMV and TMV using
a spheroid model, and it was shown that, whereas CPMV
experienced a steady diffusion profile, TMV exhibited a two-
phase diffusion behavior that entailed an extremely rapid early
loading phase, which could be attributed to its movement
axially, acting like a needle.147 Some other advantageous
properties that are conferred by elongated particles include
better margination toward the vessel wall and stronger adherence
due to greater surface area for interaction, which not only have
implications for tumor homing but also for enhanced targeting
of cardiovascular disease.148,149
Shape is a difficult parameter to account for due to the
challenge of producing monodisperse particles that can be
precisely and reproducibly tailored at the nanoscale, but this
challenge can be surmounted using VNPs and VLPs due to
the specificity of their self-assembly process. Some bottom-up
assembly approaches have been investigated recently to eluci-
date the role of aspect ratio in cell uptake, biodistribution, and
tumor homing. In one approach, CPMV particles were linked
together to form chains in order to maintain charge and surface
properties while modifying the aspect ratio, and dimers with
an aspect ratio of 2 were found to target cancer cells more
efficiently than single particles.150 Higher aspect ratios were
investigated in another study that utilized in vitro assembly of
TMV around synthetic RNA to form rods of various lengths
(300, 130, and 60 nm, corresponding to aspect ratios of 16.5, 7,
and 3.5).151 For receptor-targeted particles modified with the
RGD ligand, rods with an intermediate aspect ratio of 7 were
found to be more efficient at tumor targeting due to a combi-
nation of better macrophage avoidance and greater adhesion to
target integrins compared to the short rods and better diffusion
within the tumor compared to the long rods. Based on the
aforementioned spheroid study with TMV,147 it is likely that the
three aspect ratios experience similar axial diffusion during
the initial rapid phase, but then longer particles are impeded
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during the slower distribution phase. In contrast, PVX particles
with an aspect ratio of 40 appear not be hindered by their
length and in fact experience better penetration in relation to
icosahedral CPMV.143 Some possible reasons to account for this
include a thinner cross-section and greater flexibility for PVX
compared to TMV. To further expand understanding of the role
of shape, other factors such as density and flexibility should be
considered in future studies.
Surface presentation of shielding polymers also plays a role
in the in vivo behavior of virus-based particles. PEG is the
standard polymer used to reduce immunogenicity and non-
specific cell interactions. The importance of polymer coatings
is particularly apparent in the study highlighted above with
different aspect ratios of TMV. Although it was found that
targeted particles fared better when intermediate in size, coating
the TMV with PEG allowed the shorter rods to be better able to
avoid clearance and, paired with their superior diffusion pro-
perties within the tumor, resulted in increased passive tumor
targeting of the short 60 nm PEGylated rods.151
Surface PEGylation has been applied to many VNPs and has
been established as a broadly applicable method for extending
circulation time.102,138,152,153 Additionally, differences in the
route of clearance were also observed, with non-PEGylated
TMV and PVX filaments getting filtered through the kidneys,
while PEGylated particles do not, likely due to the increase in
the width of the particles after conjugation preventing renal
filtration.102,138,154 The conformation of the PEG coating can
be predicted computationally through estimating its surface
coverage on the particle and its hydrodynamic radius to determine
the packing density of the polymer. The use of higher molecular
weight PEG generally results in a higher hydrodynamic radius and
thus better shielding, but hydrodynamic radius is only an average
and polymers can extend and collapse in solution. Despite a
smaller predicted radius, branched PEG with multiple sites of
attachment to the particle has been shown to be more effective at
shielding than linear PEG four times its molecular weight, likely
because simultaneous tethering of the ends of the PEG traps it
closer to the particle, reducing its movement and the possibility of
nonspecific protein adsorption. We hypothesize that the branched
PEG leads to a more efficient shield, preventing the formation of
a protein corona that may tag the virus-based nanoparticles for
recognition by the innate immune system and lead to removal
from circulation (Fig. 6).102 Therefore, the dynamics of PEG in
solution should also be considered when determining its con-
formation for optimization of particle shielding.
Other polymer coatings are also being studied, with poly-
(N-(2-hydroxypropyl)methacrylamide) (pHPMA) being another
hydrophilic polymer used, particularly with AAV and adeno-
virus in order to eliminate normal infection pathways and allow
redirection of the viruses through other pathways.155,156 Some
cationic polymers, such as poly(amidoamine) (PAMAM) den-
drimers157 and polyethyleneimine,158 have also been explored
for shielding from infection as well as for improving transfection
efficiency. For more control and diversity of polymerization,
chemistries have been established for grafting polymers to
and from viral scaffolds using ATRP and ROMP, as discussed
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Fig. 6 Effect of PEG shielding on PVX clearance. (a) Diagram of conformations of PEGs of different lengths and geometries displayed on PVX based on
calculations of grafting density and Flory dimension. (b) Pharmacokinetics of the various PEGylated particles when injected in Balb/C mice show better
shielding of the 5k branched polymer. Reproduced with permission from ref. 102. Copyright 2015 Elsevier.
in Section 3.2, but their properties in vivo have not yet been
established. The use of serum albumin has been recently
investigated for coating of TMV and shown to be more effective
than medium-length PEG (5000 Da), with circulation times up
to 10-fold greater in comparison.159 Self peptides based on
human CD47 could also be considered for inhibiting phagocytic
clearance of the nanoparticles.160
Along with surface modifications that allow them to avoid
undesirable cell interactions, particles can be enhanced for
specific cell targeting through the display of receptor-specific or
disease environment-specific ligands. Some examples of targets
that have been used for specific uptake of virus-based nano-
particles include epidermal growth factor receptor (EGFR)161,162
and folate receptor (FR).163,164 In such a manner, overexpressed
receptors or environmental cues can be tracked for diagnostic
or drug delivery purposes, which will be discussed in the
following sections. To obtain the most favorable ligand display
density, there is a balance between increasing avidity and
reducing cellular receptor depletion that arises from increased
ligand density.165 While multivalency and a higher degree of
labeling with targeting ligands is beneficial for stronger cellular
interactions, too many ligands may reduce the extent of
endocytosis through exhaustion of cellular receptors. Another
design parameter for the inclusion of targeting ligands is the
linker used for attachment. For example, the inclusion of PEG
can assist in increasing circulation time and avoiding non-
specificity as discussed above. Additionally, PEG linkers can
improve cell targeting by adding flexibility and enhancing
presentation of targeting peptides.166 By altering the character-
istics of the linker, the interaction of the ligand with its target
and the overall behavior of the particle in vivo can be tuned.
4.1.2 Imaging. Viruses have been used for tissue-specific
imaging and delivery of contrast agents in applications of
optical imaging, magnetic resonance imaging (MRI), and positron
emission tomography (PET). The utility of using viruses as imaging
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probes comes from the diversity of approaches for modification
of the particles as well as the ease of precise assembly. In
addition, clearance and removal from the body are critical for
preventing toxicity from tissue retention of contrast agents,
and many VNP platforms tend to be cleared quickly from the
body (half-life of minutes)137–139,154 compared to some synthetic
materials that require months for clearance, such as carbon
nanotubes, gold, and silica.167–169 Imaging is an important tool
in medicine for diagnostics and for visualization of disease
localization and progression, as well as treatment success. With
improvements in imaging technology, earlier disease detection
and better prognosis can be realized. The ability to track particles
further aids in the evaluation of drug delivery platforms, as it can
be used for confirmation of cell-specific uptake and investigation
of interactions of particles within the body, such as their
clearance, biodistribution, and immunogenicity.
Fluorescence imaging is the main modality for preclinical
evaluation and was used to aid in the establishment of the
design rules in Section 4.1.1. Fluorescent agents can be incor-
porated into viral capsids through bioconjugation,170,171 genetic
engineering,84,86 infusion,110,114 and self-assembly.21,130 Fluores-
cence is useful for quantification of particle uptake using flow
cytometry, visualization of particle localization through confocal
microscopy, and determination of biodistribution using in vivo
imaging. Although high dye densities can easily be achieved
through efficient capsid modification strategies, sensitivity
decreases after a certain threshold due to fluorophores experi-
encing quenching when placed at distances less than approxi-
mately 10 nm from each other. Therefore, a fairly low density of
around 10% is more ideal for achieving optimal fluorescence
intensity.172 Encapsulation of indocyanine green can be utilized
as a method for near infrared (NIR) photoacoustic imaging, and
it has shown greater photostability compared to the chromo-
phore alone.173 As advancements are made, another aspect that
could be explored is the integration of gold nanoparticles with
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fluorophores for metal-enhanced fluorescence with improved
quantum yields and decreased photobleaching.174
First iteration native and PEGylated particles can be directly
evaluated for their biodistribution, pharmacokinetics, and tumor
homing behavior through fluorescence imaging.84,90,102,138,143,175
Overall, particles are cleared mainly through the liver and spleen,
with filamentous particles having a higher rate of spleen
clearance compared to icosahedral particles,138,143 localizing
with B cells within the white pulp over time.102,138 Due to leaky
vasculature and poor lymphatic drainage, the enhanced perme-
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ability and retention (EPR) effect is found in solid tumors and
can be utilized for tumor imaging through nanoparticle deposition.
Using both mouse and chicken chorioallantoic membrane (CAM)
models with tumor xenografts, the passive partitioning of particles
to the tumor can be observed (Fig. 7).84,143 As discussed in the
previous section, evaluation of localization of particles inside
the tumor revealed enhanced accumulation and penetration of
rod-shaped particles.143
Besides passive tumor homing properties, natural interactions
of viruses with certain cells can also be exploited. CPMV in
particular exhibits unique specificity in interacting with surface
vimentin, which is found on endothelial, cancer, and inflamma-
tory cells.176–179 The native affinity of CPMV for surface vimentin
allows for high-resolution imaging of microvasculature up to
500 mm in depth, which cannot be achieved through the use of
other nanoparticles, as they tend to aggregate and block the
vasculature.180 This interaction can be utilized for a range of
applications, such as delivery to a panel of cancer cells including
cervical, breast, and colon cancer cell lines,110 delineation of
atherosclerotic lesions,177 and intravital imaging of tumor vascu-
lature and angiogenesis.180 Another example of an existing endo-
genous association is CPV with transferrin receptor (TfR), an
important receptor for iron transport into cells and highly
upregulated by numerous cancer cell lines.52 Even after dye
labeling, CPV retains its specificity for TfR and was shown to
bind to receptors found on HeLa cervical cancer cells, HT-29
colon cancer cells, and MDA-MB-231 breast cancer cells. As a
quick side note, in vivo imaging of bacterial infections and
differentiation between F-positive and F-negative E. coli strains
is also possible through specificity of binding of M13 phage.181
Specificity can also be introduced through the incorporation
of targeting ligands for molecular imaging. RGD is a targeting
peptide that is frequently used due to its high affinity for avb3
and avb5 integrins, which are involved in angiogenesis and
associated with cancer proliferation.81,151,182,183 The association
of RGD-targeted particles with tumor vascular endothelium has
been demonstrated in mice, although the study also indicated
that better tumor localization would be achieved with greater
circulation time imparted through incorporation of a better
shielding linker (see Section 4.1.1).151 CPMV displaying peptide
F56, which was discovered through phage display, has been used
to target vascular endothelial growth factor receptor-1 (VEGFR-1),
with accumulation throughout the tumor observed compared to
no detectable uptake of non-targeted particles.99 Other options
that have been explored include FR targeting with folic acid
(FA),164,184,185 TfR with transferrin,186 and prostate specific
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membrane antigen (PSMA) with a PSMA antibody.187 In recent
years, a target that has been approached from many angles is
epidermal growth factor receptor (EGFR), an important bio-
marker overexpressed on many malignant cell types. Strategies
range from display of EGF on Qb through genetic engineering,162
using phage antibody libraries to select for fd phages with single-
chain antibody variable fragments (scFvs) specific for EGFR as
well as its related receptor human epidermal growth factor
receptor 2 (HER2),101 conjugation of EGFR antibodies on
MS2,188 and also chemical attachment of GE11 peptide on
PVX.161 These studies all evaluated cell binding in vitro and
there are some promising results indicating partitioning of
targeted particles to tumor cells compared to macrophages in
co-cultures,161 and it would be of interest to see their development
in mouse models.
Outside of membrane proteins of cancer cells, proteins
highly expressed by activated endothelial cells, such as vascular
cell adhesion molecule (VCAM)-1, can be utilized for targeted
imaging of cardiovascular disease and atherosclerotic plaques.148
Beyond such strategies, matrix and secreted proteins are also
advantageous targets. For example, collagen189 and secreted
protein acidic and rich in cysteine (SPARC),190,191 an extra-
cellular matrix glycoprotein, can be detected for tumor imaging
through target-specific peptides displayed on M13. SPARC in
particular has been successfully targeted for deep tissue imaging
of lung cancer,191 and it has even been used for guided resection
of ovarian cancer through the pairing of M13 with fluorescent
single-walled carbon nanotubes (SWNTs),190 which allows for
non-photobleached fluorescence with less background in the
second NIR window ranging from 950–1400 nm. The poly-
merized fibrin found in thrombi has also been investigated
for the delineation of blood clots using MS2, CPMV, and TMV
equipped with GPRPP149,192 and CREKA149 pentapeptide amino
acid sequences.
The above studies investigating imaging of thrombosis149
and atherosclerosis148 established target-specific imaging not
only with optical but also with MR imaging. MRI is a clinically
relevant method for noninvasive disease characterization with
good soft tissue contrast, and the use of contrast agents in
conjunction with MR can improve the signal-to-noise ratio to
highlight differences between diseased and normal tissues.
Gadolinium (Gd) is one such paramagnetic contrast agent that
can be used to achieve brighter signal in T1-weighted imaging.
Molecular imaging of atherosclerotic plaques was achieved at
dosages 400 times lower than clinically used for angiography
with the encapsulation of Gd chelated with 1,4,7,10-tetra-
azacyclododecane-1,4,7,10-tetraacetic acid, or Gd(DOTA), within
TMV targeted to VCAM-1.148 The high contrast can be attributed
to the high payload of 1200 Gd/TMV, the slower molecular
tumbling rate resulting from attachment of Gd(DOTA) to the
macromolecule, inclusion of the targeting peptide, and the
advantage imparted by the shape of TMV for drifting laterally
to the vessel wall.
This latest result is the culmination of numerous studies by
several groups that formed stepping stones along the way.
While early work looked at the direct binding of Gd to natural
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Fig. 7 Imaging of tumor uptake and distribution of CPMV and PVX. (a) Comparison of icosahedral CPMV (green) and filamentous PVX (red) distribution
when coinjected in a CAM model of chick embryos prepared with vascularized GFP-expressing human fibrosarcoma HT1080 or human epithelial
carcinoma HEp3 tumors (magenta), with PVX better able to penetrate to the tumor core. Scale bar = 190 mm. (b) Fluorescence microscopy of 8 mm tumor
sections showing CPMV having limited distribution, while PVX is spread throughout the tumor and found in areas devoid of CPMV (white arrowheads).
(c) Image of tumors from an HT-29 colon cancer mouse xenograft model after intravenous injection of CPMV and PVX particles (left) and quantitation of
fluorescence intensity (right). (d) Immunofluorescence staining of 10 mm tumor sections showing CPMV (pseudocolored in yellow) remaining close to the
endothelium (stained with FITC-labeled CD31 antibody pseudocolored in pink) and PVX (pseudocolored in green) having better tissue penetration
properties. Nuclei were stained with DAPI (blue). Scale bars are 30 mm. Reproduced with permission from ref. 143. Copyright 2012 American Chemical
Society.

metal binding sites in the capsid of CCMV,193 the use of
chelation and bioconjugation was quickly introduced to mitigate
concerns of free Gd leading to toxicity in patients with underlying
kidney disease, with explorations using both DOTA93,194 and
diethylenetriaminepentaacetic acid (DTPA).195 These approaches
resulted in per particle T1 relaxivities on the order of 1000 to
8000 mM1 s1 measured at 64 MHz. While fairly high and
much greater than 20 mM1 s1 for Gd alone, these values do
not approach the measurement of around 28 000 mM1 s1 per
particle from the initial study with direct attachment of Gd.193

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To improve contrast, interior and exterior labeling196,197 as well
as rigidity of linkers198 was explored with MS2 using chelators
based on hydroxypyridinonate (HOPO) due to its 3-fold relaxivity
enhancement compared to clinically used Magnevist, or
Gd(DTPA). Internally modified capsids and more rigid linkers,
in particular the S,S enantiomer of 1,2-cyclohexyldiamine, each
demonstrated approximately 30 to 40% higher relaxivities.
Enhancing Gd loading was another method explored to
increase per particle relaxivity, either using ATRP to amplify
density of groups with which to attach the contrast agents104,105
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or using branched oligomers with multiple Gd(DTPA) com-
plexes attached.199,200 The greatest success with this approach
led to the incorporation of over 9000 Gd(DTPA) per P22 particle,
with per particle relaxivities exceeding 200 000 mM1 s1.104
In vivo imaging was first demonstrated using P22 conjugated to
Gd(DTPA) in order to visualize blood vessels in a mouse, with
clear depiction of the carotid artery, mammary arteries, the
jugular vein, and veins in the head at a resolution of 250 mm.200
More recently, relaxivities approaching 1 000 000 mM1 s1 per
particle were reported by utilizing TMV’s greater surface area
conferred by its shape to introduce a large payload of Gd(DOTA),
accompanied by thermal transition of the rods using conditions
that result in 200 nm spheres.201 Coating interiorly labeled TMV
particles with silica could potentially increase the relaxivity
3-fold as well as lead to greater macrophage uptake and hence
contrast.202 It is expected that a combination of these research
directions investigating chelators, linkers, conjugation, shape
shifting, and coating will result in particles with even greater
contrast for better visualization of disease.
Apart from Gd-based contrast enhancement, manganese and
iron oxide are other contrast agents that have been investigated.
Manganese research is relatively new and labeling of P22 with
Mn porphyrins was shown to have a per particle relaxivity of
7000 mM1 s1 at 90 MHz, and while this is low compared to
advancements in Gd imaging, it is a promising avenue to pursue
due to the reduced toxicity of Mn.203 Unlike Gd and Mn, iron
oxide is a contrast agent for T2-weighted imaging and is observed
from a resultant darker image. Interestingly, the first demonstra-
tion of MR imaging with iron oxide was in plants, where cubic
Fig. 8 Targeted MR imaging of prostate cancer with M13. (a) Diagram of M13 structure with the major p8 proteins displaying a triglutamate motif for the
multivalent display of iron oxide nanocrystals (black circles) and the p3 proteins at the end of the virus displaying SPARC binding peptide (pink) for
targeting. (b) MR scans of mice using a 7 T small animal MR scanner with subcutaneous C4-2B tumors (encircled) before (left) and 24 hours after (right)
M13 injection displayed dark contrast from the targeted particles against the bright image of the tumor. Reproduced with permission from ref. 205.
Copyright 2012 Nature Publishing Group.
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iron oxide nanoparticles were encapsulated within BMV, Nicotiana
benthamiana leaves were infiltrated with the modified virus, and
imaging was performed with cell-to-cell trafficking of the encap-
sulated iron oxide observed.204 Encapsulation of iron oxide nano-
particles and phantom imaging has been demonstrated with
hepatitis B core VLPs.77 Moving toward translation, attachment
of iron oxide nanoparticles along with SPARC binding peptides
to the surface of M13 was effective for the imaging of prostate
cancer (Fig. 8).205
Another MR contrast approach that is quite new is chemical
exchange saturation transfer (CEST) and hyperCEST imaging.
Xenon-based agents in particular have been explored for
viruses. After selective saturation of these nuclei, an enhanced
water signal is observed due to saturation transfer to surrounding
bulk water. This technique has found success with MS2,206
M13,207 and fd,208 with sensitivities as low as 230 fM.207 By
additionally incorporating scFVs that recognize EGFR, molecular
imaging and contrast specificity were demonstrated with MDA-
MB-231 cancer cells, with essentially no contrast in Jurkat negative
control cells.208 Due to the more than 10 000-fold increase in
sensitivity, there is a lot of potential in this new technology.
PET imaging is another sensitive imaging modality and relies
on the detection of radiotracers. It has been utilized for ascer-
taining the biodistribution of non-PEGylated209 and PEGylated210
MS2 capsids through incorporation of [18F]fluorobenzaldehyde
and 64Cu chelated with DOTA, respectively. Taking it a step
further, biodistribution of encapsulated or non-encapsulated
superparamagnetic iron oxide nanoparticles, 18F, and poly-
18
L-lysine (PLL) cation (for packaging F) within hemagglutinating
virus of Japan envelopes (HVJ-Es) was studied to determine
whether magnetic stimulus can be used to redirect the viruses
to the head, and it was clear that the application of the magnets
altered the fate of the viruses, with increased signal in the
head.211 Targeting ligands were also explored in conjunction
with PET imaging, with RGD for targeting human sarcoma212
and glioma213 xenografts and GE11 for targeting an EGFR
positive liver cancer xenograft model.157 In the context of virus-
based particles, there has been less work with PET compared to
the other imaging modalities. While PET has its advantages of
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high sensitivity and ability to image more deeply, radiotoxicity is
an issue. For the purposes of simply detecting particle localization
for diagnostics, MRI may be more ideal to pursue as technology
improves.
4.1.3 Vaccines and immunotherapy. We will begin our
foray into viral vectors for combatting diseases starting with
vaccines, which has an extensive history and is likely the first
medical application of viruses. Its popularization had an
illustrious beginning in 1796 with Edward Jenner’s experiment
inoculating his gardener’s eight-year-old son with cowpox,
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which resulted in protecting the boy from subsequent challenges
with the more serious smallpox virus.214 While knowledge of
viruses would not come until 100 years later, with the work of
Dmitry Ivanovsky and Martinus Beijerinck filtering TMV from
plant sap and demonstrating its infectivity and replication,7 the
medical application of viruses had its roots here.
Instead of live viruses, safer alternatives for vaccines have
since been established, including attenuated viruses, inactivated
or subunit viruses, non-infectious VLPs, nanoparticle delivery, and
nucleic acid vaccines.215,216 Vaccines have been researched for a
wide range of diseases, with great success for some diseases such
as polio217 and measles,218 but some important vaccines such as
for HIV and EBOV are still lacking, which are discussed below.
Eliciting an effective and long-term immune response is one
challenge for vaccines, and the use of virus capsids offers the
advantages of multivalent antigen presentation, incorporation
of multiple epitopes, and particle stability. Since the field is
enormous, we would like to feature just some of the research on
vaccination, focusing on a few studies in the areas of infectious
disease, brain disorders, and cancer. For a more comprehensive
overview of virus-based particles for vaccines, the reader is invited
to consult further reviews.219,220
In the realm of infectious diseases, HIV is particularly
challenging to address due to sequence diversity and difficulty
in generating broadly neutralizing antibodies. This is likely to
be partially due to its structural characteristics, consisting of
a low number of envelope spikes that allows it to escape
recognition as foreign and increases the likelihood of evolving
envelope determinants that mimic self.221 High density display
of HIV antigens is one method to combat this, with trimeric
glycoproteins gp41 and gp120, as well as their precursor gp160
being highly targeted. As an example, a recent investigation
studied the effect of presentation of the particularly conserved
membrane-proximal external region (MPER) of HIV-1 gp41 on
VLPs, and the approach produced anti-MPER antibodies that
showed neutralizing activity in a rabbit model.222 Albeit moderate,
the production of neutralizing antibodies is a valuable therapeutic
response that could be improved through modifying MPER pre-
sentation. Another potential target for vaccines is CCR5, a cellular
self-protein found to be involved as a co-receptor for HIV replica-
tion and pathogenesis. High-density display of CCR5-based
peptides on Qb resulted in high IgG antibody titers, which was
able to protect 25% of rhesus macaques against intravaginal
challenge with the highly virulent SIVmac251 strain.223 In terms
of potential vaccines undergoing clinical trials, some success
has been seen for the use of a treatment combining ALVAC,
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a canarypox vector vaccine, with boosters of AIDSVAX, a gp120
subunit vaccine, where vaccine efficacy of 31.2% was observed
in a study consisting of 16 395 subjects in Thailand.224 While
work still remains to be done to improve the efficacy of HIV
vaccines, great strides have been made in recent years toward
its realization.
Another area where vaccine production is of great interest
is for protection from the highly virulent and deadly EBOV.
Ebola VLPs have been generated consisting of glycoprotein,
nucleoprotein, and VP40 matrix protein from the virus using
a baculovirus expression system, and cynomolgus macaques
vaccinated with the VLPs were completely protected against
lethal EBOV challenge, with strong T cell responses likely
contributing.225 Further investigation of Ebola VLPs consisting
of glycoprotein and VP40 also produced in insect cells demon-
strated the potential of delivery without adjuvants and revealed
a strong immune response that protected against lethal challenge
in mice when high doses (50 mg) were utilized.226 Optimization is
still needed to enhance immunogenicity, and some prospects
include improving glycoprotein incorporation during VLP assembly
and including immunostimulatory molecules within the particles.
A potential safer alternative to Ebola VLPs is the use of other viruses
to display EBOV antigens instead. Vesicular stomatitis virus (VSV) is
one such virus that has been studied, and using highly attenuated
forms of VSV that have been genetically engineered to incorporate
EBOV glycoprotein in place of its own has been an effective strategy,
with a single dose being sufficient to protect both guinea pigs and
macaques from challenge.227 These results are highly encouraging,
and it will be interesting to see if there is effective protection
against other strains of EBOV.
Aside from vaccines for human viral infections, it is of great
interest to investigate animal vaccines as well for the protection
of pets and livestock. An early study inserted a short epitope
from the VP2 capsid protein of mink enteritis virus (MEV)
within the capsid of CPMV and found that it imparted protec-
tive immunity against clinical disease in mink, with a dose of
1 mg not only offering complete protection but also reducing
shedding of the virus.79 Since the epitope occurs in canine
parvovirus and feline panleukopenia virus as well, the same
platform could be used for protection of minks, dogs, and cats.
As another example, foot-and-mouth disease virus (FMDV) is a
highly infectious virus that affects cloven-hoofed animals such
as cattle and sheep, which are important in farming. Empty
FMDV capsids produced using a baculovirus system and tested
in guinea pigs were able to generate neutralizing antibodies
against FMDV, but at a lower level than the commercial
inactivated vaccine.228 There was still good antigenicity and
immunogenicity, and use of crude protein extracts may have
resulted in lower particle quantities in the experimental setup.
Thus, the results are exciting for the use of noninfectious empty
capsids to treat FMDV. Bluetongue virus (BTV) is another
problematic disease that has been detrimental to the agricultural
industry due to its high morbidity and mortality, affecting
ruminants such as sheep and cattle. VLPs of BTV produced
using a baculovirus expression system were found to be protective
against infection when tested in sheep, with effective delivery of
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both single-serotype and multi-serotype cocktails and no inter-
ference observed from the presence of antibodies against other
serotypes.229 BTV VLPs can also be assembled in plants using a
CPMV-based HyperTrans vector system, and vaccination with
these VLPs provided protection against BTV challenge.230 The
particles were found to elicit a strong antibody response in sheep
after a booster dose, comparable to live, attenuated virus used in
a commercial vaccine. The development of vaccines in plant-
based systems could result in cheap, easily scalable production
without the danger of animal pathogen contamination.231
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Interestingly, vaccines can also be applied to brain disorders
such as addiction and Alzheimer’s disease. Nicotine from
tobacco use is the most common drug addiction worldwide,
and reduction of nicotine transport to the brain has been found
to decrease dependency on the drug due to reducing stimula-
tion of the mesolimbic reward system.232 Nicotine covalently
coupled to Qb resulted in high drug-specific IgG antibody
production in vaccinated mice, and the binding of the anti-
bodies to nicotine caused a decrease in nicotine levels in the
brain of up to 90% in individual mice.233 Further, Phase I trials
found that this approach was safe and well tolerated, with high
antibody production against nicotine in all individuals. Phase
II trials have demonstrated that nicotine vaccines can be
effective for patients quitting smoking, but there is still room
to improve antibody titers for greater efficacy.234
Turning to Alzheimer’s disease, amyloid-b (Ab) peptide
deposits are associated with the development of the neuro-
degenerative disorder and can be targeted with vaccines to
reduce aggregation and ameliorate symptoms. In one study,
hepatitis B virus core proteins displaying two 15-amino acid Ab
fragments taken from the N-terminus were assembled into
chimeric VLPs and used in the immunization of an Alzheimer’s
transgenic mouse model without inclusion of additional
adjuvant.235 The VLPs elicited a potent humoral response that
reduced Ab deposition and microgliosis, and there was a resultant
improvement in learning and memory, with immunized mice
more readily learning and remembering the location of the
hidden platform in a Morris water maze. Another therapeutic
that has potential for Alzheimer’s disease is CAD106, a
Qb-based vaccine displaying the first six amino acids of Ab
that is undergoing Phase II testing for long-term treatment of
patients with mild Alzheimer’s disease.236 Overall, multiple
exposures to CAD106 resulted in a prolonged time to antibody
titer decline, and the treatment had favorable safety and tolerability
profiles, with no occurrences of the severe adverse responses
found in Ab antibody therapies, such as meningoencephalitis
and autoimmune disease. More time is needed to observe the
long-term treatment effects of the vaccine, but so far the results
are promising.
Finally, we look at the treatment of cancer through a few
different immunotherapy approaches. Recently, Merck brought
Gardasil 9 to market, an HPV vaccine upgrade from Gardasil
that protects against 9 serotypes of HPV that account for 90% of
HPV-related cancers.237 While this is fantastic news, alterna-
tives are necessary due to issues with cost and distribution in
the developing world. Commercial vaccines are VLPs derived
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from the L1 major capsid protein of HPV, which is not conserved
across serotypes. Thus, a simpler approach that can be more
broadly protective would be more ideal. As an example, efforts
have been made to produce highly immunogenic L2 VLPs that
are stable over time without refrigeration.238 Although the L2
minor capsid protein of HPV is less exposed and less immuno-
genic, it is highly conserved across serotypes, and MS2 displaying
a short L2 peptide from HPV type 16 worked particularly well in
preclinical mouse models. Reconstituted virus after spray drying
remained highly immunogenic without use of an adjuvant even
after 7 months of storage at room temperature, and mice
vaccinated with the 16L2-MS2 VLPs were additionally protected
from heterologous HPV pseudovirions of types 31 and 45, while
Gardasil only protected against type 31.
Vaccines can also be used for protection by stimulating the
immune system against the cancer cells themselves. HER2 is a
receptor overexpressed on breast cancers that tend to be more
aggressive and is one potential target for cancer immunotherapy.
Presentation of P4378–394, a B-cell epitope from the extracellular
domain of HER2, on PVX led to higher antibody titers that were
specific to HER2 compared to soluble P4 peptide alone.239 PVX-
based carriers are promising for vaccines due to their tropism
toward B cells154 and large surface area imparted by their
filamentous nature leading to high multivalency. A different
approach against HER2 cancer cells resulted in T cell mediated
tumor prevention by utilizing the association between the
minor capsid protein VP2 of murine polyomavirus (MPyV) with
the internal surface of major capsid protein VP1.240 The VP2
coat proteins were fused to HER21–683 and the particles were
assembled in such a way that immunization of mice with the
VLPs resulted in antibodies to VP1 but not HER2. On the other
hand, the VLPs induced the production of HER2-specific T cells,
and in both an autochthonous HER2 breast cancer mouse model
and a model in which mice were challenged with HER2-positive
D2F2/E2 cells (but not in a model with HER2-negative D2F2
cells), complete protection against tumor growth was observed
for over 80% of the mice. Therefore, this is a potential approach
for a potent prophylactic vaccine against HER2 cancer. Beside cell
surface receptors, Tn antigen, a tumor-associated carbohydrate
antigen, has been widely explored for presentation on viral
capsids, with investigations into utilizing particles such as CPMV,
TMV, and Qb.241–243 Although Tn is one of the weakest antigens,
strategic patterned display of the antigen was able to induce a
potent humoral immune response that recognized human tumor
cells. Evaluation of Tn presentation for in vivo cancer protection
will be an essential next step for carrying this forward as a cancer
vaccine therapeutic.
It is clear that by taking advantage of the body’s natural
immune system, myriad applications for virus-based vaccines
can be realized. We would like to briefly highlight the application
of viruses for immunotherapy as well, especially with the recent
groundbreaking approval of the first oncolytic virus (OV) for
cancer therapy by the FDA in October 2015.135 T-VEC was
approved for the treatment of melanoma patients and is a viral
vector based on HSV type 1. The mechanism of action for OVs is
not yet well understood but appears to involve both lysis from
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more rapid replication within tumor cells as well as promotion
of systemic anti-tumor immune response.244 T-VEC encodes for
granulocyte-macrophage colony-stimulating factor (GM-CSF),
a cytokine involved in dendritic cell recruitment maturation
that aids in additional stimulation of anti-tumor immunity. In
phase III clinical trials, treatment with T-VEC led to durable
responses, even for patients with advanced stage IV disease.
The approval of T-VEC is a significant step forward, and future
investigations will be important for enhancing efficacy through
combination approaches (Fig. 9) as well as for the establish-
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and infectious diseases, progress is being made and recent
results show great promise in the efficacy of some novel vaccines.
The potential of VLPs for vaccines and immunotherapies is quite
evident, with opportunities for the treatment of not only infectious
diseases but also addiction, brain disorders, cancer, and more.
4.1.4 Gene delivery. In addition to vaccines, viruses have
been applied for the delivery of full-length DNA, small inter-
fering RNAs, and machinery for genome editing, such as
nucleases, for treatment of a wide range of disorders. Gene
delivery has had a long history, beginning with its initial

ment of safety profiles and regulatory guidelines. For more
information on the action of OVs and other recent developments
in this field, please refer to the following reviews.244,245
As a final example of the effectiveness of virus particles for
immunostimulation, a recent study, in which we collaborated
with the Fiering Lab at Dartmouth college, demonstrated that
in situ vaccination of tumors with eCPMV, just the capsid
without any nucleic acid or modification, could help overcome
the immunosuppressive tumor microenvironment.246 Treatment
with eCPMV led to reduction and even regression of tumor
growth and metastasis in a variety of mouse models, including
melanoma, ovarian carcinoma, colon cancer, and breast
carcinoma. eCPMV was found to specifically target and activate
neutrophils in the tumor microenvironment, leading to a
strong and rapid anti-tumor response. The response was found
to be systemic and durable, with mice that eliminated primary
B16F10 melanoma tumors through eCPMV-mediated immunity
resistant to re-challenge and three out of four mice completely
rejecting the tumor (Fig. 10). Systemic protection is likely the
result of immune memory against tumor antigens and mediated
by T cells. These results were established for unmodified eCPMV,
therefore opening the opportunity for further enhancement
of efficacy through the display of antigens or the inclusion of
adjuvants and chemotherapeutics.
Overall, while there are still difficulties that exist for the
development of vaccines targeting chronic diseases, cancer,
conceptualization over 40 years ago.247 Since then, there has
been an extensive body of work dedicated to the realization
of gene therapy for treatment of diseases, with Parkinson’s
disease,248 cystic fibrosis,249 hemophilia,250 and cancer251 being
just a few examples. The extension of gene therapy for the
general public is starting to become more realistic, with the
approval of UniQure’s Glybera for clinical use in Europe in 2012
opening the door for bringing gene therapies into the market.252
The AAV therapy delivers the lipoprotein lipase gene to make up
for deficiency in patients who cannot process triglycerides. Other
gene therapies are poised to push toward commercialization in
the coming year, among which includes an AAV gene therapy
from Spark Therapeutics for the treatment of Leber congenital
amaurosis visual impairment, which is caused by defects in the
RPE65 gene.253 Based on lack of serious adverse events from
initial studies and positive phase III trial results, they are
expected to advance toward filing a Biologics License Application
with the U.S. Food and Drug Administration.
The road to this point has certainly not been easy, with
tragedy striking in 1999 when Jesse Gelsinger died during a
clinical trial from a massive immune response instigated by the
adenoviral vector meant to correct for his ornithine trans-
carbamoylase metabolic deficiency.254 Only a few years later,
despite great success for many children, retrovirus-based gene
treatment for X-linked severe combined immunodeficiency
resulted in the development of leukemia in several young

Fig. 9 Oncolytic virus therapy action and potential synergy. (a) Immune clearance of tumors at baseline is inhibited by inactivation of T cells through
binding of their programmed cell death protein 1 (PD1) receptor to programmed death ligand 1 (PDL1) expressed on tumor cells as well as by secretion of
inhibitory cytokines. (b) OV treatment triggers local expression of pro-inflammatory cytokines and/or overrides immune checkpoint inhibition, resulting
in immune stimulation and recruitment of immune cells. (c) Combination of OV therapy with other immunotherapies such as PDL1 antibodies and
chimeric antigen receptor-expressing T cells can be used to enhance immune responses. Reproduced with permission from ref. 245. Copyright 2015
Nature Publishing Group.

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Fig. 10 Systemic anti-tumor immunity after in situ vaccination with eCPMV. (a) Images of mice with flank intradermal B16F10 melanoma tumors three
days after intratumoral injection of either eCPMV or PBS demonstrate slower growth with eCPMV. (b) Tumor measurements of the mice after treatment
(arrows indicate treatment days), with significant decrease in tumor progression rate for eCPMV (n = 8 for eCPMV, n = 6 for PBS). (c) Kaplan–Meier curves
illustrate survival of half of the mice treated with eCPMV, with complete elimination of primary tumors observed for those mice. (d) Rechallenge on the
opposite flank 4 weeks later (n = 4/group) also saw delayed growth for eCPMV, and 3 out of 4 mice did not develop new tumors. *p o 0.05; **p o 0.01;
***p o 0.001. Reproduced with permission from ref. 246. Copyright 2015 Nature Publishing Group.

children as a result of gene insertion near oncogenes.255 Despite
these misfortunate and discouraging results in the past, more
research has led to a better understanding of gene delivery, its
potential pitfalls, and how to overcome them, leading to a lot
more control over accomplishing the purpose of gene delivery
with less severe and fewer adverse effects. Due to the plethora of
work done in this area, we can by no means be comprehensive in
our review of gene delivery, and there are many excellent reviews
that we invite the reader to consult.256–258 However, we will take a
glimpse at a few studies to demonstrate the breadth of the field
and the exciting studies being performed. We will then focus
more on some recent developments in improving the safety
profile of adenovirus and retargeting it for cell-specific delivery
as well as the progress in utilizing non-mammalian viruses for
gene delivery.
Adenovirus is an icosahedral non-enveloped virus with a
core diameter of 90 nm and fibers that extend from its penton
bases, which allow attachment to the host cell through the
coxsackie-adenovirus receptor (CAR) (see Fig. 1).259 Following
binding, adenovirus can further bind to integrin receptors
through RGD displayed at the base of the fibers. The specificity
of adenovirus for mammalian cells can be utilized for gene
therapy for cases such as glioblastoma, a highly aggressive
brain cancer, with the ability to prolong time to death or
reintervention.260 This tactic works by injecting adenovirus
containing cDNA for herpes simplex virus thymidine kinase
(HSV-tk) around the lesion after surgical resection of the tumor.
Following systemic delivery of the prodrug ganciclovir, cells
that are transduced with HSV-tk can then phosphorylate the
prodrug to ganciclovir triphosphate, a cytotoxic nucleotide
analogue that becomes incorporated into the DNA of actively
proliferating cells. Adenovirus gene delivery can also be utilized
as an antiviral therapeutic through RNA interference (RNAi),
and this has been demonstrated for HBV using delivery of
anti-HBV primary microRNA (pri-miR) cassettes driven by a
murine transthyretin (MTTR) promoter.261 The promoter is a
liver-specific transcription regulatory element and imparted
high specificity, with barely detectable expression in non-liver
cells. Furthermore, expression of anti-HBV pri-miRs resulted in
knockdown of HBV replication up to 94% in mice.
As an alternative to adenovirus delivery, AAV is particularly
popular due to its very low immunogenicity, inability to self-
replicate, and capacity to target non-dividing cells.262 Stereo-
tactic delivery of nerve growth factor (NGF) into nucleus basalis
neurons in the basal forebrain has been shown to be a well-
tolerated method for long-term NGF expression for the purposes
of protecting patients with Alzheimer’s disease from neural
degeneration.263 AAV vectors can additionally provide protection
from simian/human immunodeficiency virus (SHIV).264 Through
delivery of a transgene encoding for eCD4-Ig, which binds to the
CD4 envelope glycoprotein of HIV-1 as well as co-receptor CCR5
with high avidity, protection from multiple infectious doses of
SHIV was conferred in rhesus macaques. AAV/phage hybrids
(AAVP) have also been created in order to combine the targeting
potential of phages with the transgene expression efficiency of
AAV by inserting cis-regulatory elements from AAV into the M13
phage vector genome flanking the transgene cassette.212 Using
the HSV-tk and ganciclovir approach described above for adenovirus,

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AAVP displaying RGD peptides was able to home to sarcoma cells
and instigate transgene expression and tumor regression in a rat
SKLMS1 human soft-tissue sarcoma xenograft model.
A similar outcome for combining targeting and transfection
can also be achieved through covalent coating of adenovirus
with a hydrophilic polymer to result in retargeting of the virus
as well as protection from antibody neutralization.155 High
immunogenicity is one of the downfalls for adenovirus delivery,
resulting mainly from intravascular injection,265 and anti-
bodies produced are primarily against the hexon capsid protein.266
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While adenovirus can still be utilized for local administration, by
shielding the hexons and incorporating targeting ligands, systemic
delivery with target selectivity independent of CAR is made
possible. Amine-reactive pHPMA-based polymers (mentioned
previously in Section 4.1.1) have been used for adenovirus
coating, and further modification of the virus with targeting
ligands such as basic fibroblast growth factor (bFGF) and
vascular endothelial growth factor (VEGF)155 or antibodies against
E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) fused to
IgG1 Fc267 have been demonstrated for retargeting of adenovirus
to cancer cells and tumor-associated vasculature. Additionally,
pHPMA-coated adenovirus with activatable cell penetrating
peptides attached enabled cytoplasmic delivery of the virus to
metalloproteinase-overexpressing tumor cells.268 Transduction
with pHPMA copolymer coatings was shown to be maintained
not only in cell cultures but also in vivo in mouse models.267,269
PEG has also been shown to be an effective polymer for shielding270
and retargeting.271 As an improvement to simple PEG coatings,
complexation with copolymers of PEG and PEI resulted in the
ability to transduce CAR-negative NIH 3T3 cells, with the added
benefit of less toxicity compared to PEI alone.272 Aside from PEI,
cationic poly(amidoamine) (PAMAM) dendrimers can also be used
for enhancing gene delivery. Through the addition of EGFR-
specific ligand GE11, dendrimer-coated adenovirus carrying the
sodium iodide symporter gene (NIS) was successfully applied for
radiovirotherapy in a liver cancer xenograft mouse model, in
addition taking advantage of iodide for 124I PET imaging of viral
distribution.157
While mammalian viruses have the machinery for gene
transduction, phages and plant viruses also hold potential for
gene delivery and gene silencing. Early phage gene delivery was
reported through targeting M13 to EGF with a mammalian gene
cassette inserted into the vector backbone.273 Transduction was
low but could be improved through the use of multivalent
phagemid-based vectors (discussed in Section 4.2.1) and geno-
toxic treatment such as heat shock, UV irradiation, and campto-
thecin treatment. As a more practical method to overcome
cellular barriers of mammalian cells, inspiration from HBV
was taken and the PreS1 region of the HBV envelope protein
involved in virus attachment during infection was displayed on
bacteriophage T7, which resulted in more efficient gene transfer
when tested in HepG2 human hepatocellular carcinoma cells.274
Another hybrid phage complex, this time with cationic polymers
poly-D-lysine (PDL) and diethylaminoethyl-dextran (DEAE-DEX),
was also able to improve transgene expression, and cell type
specificity was retained from the display of RGD.275 Gene delivery
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with plant viruses is only beginning to take shape and has recently
been demonstrated through assembly of CCMV coat proteins
around heterologous RNA derived from Sindbis virus (SINV),
which was shown to be released into the cytoplasm of mamma-
lian cells through co-delivery of Lipofectamine-2000 (Fig. 11).276
For gene silencing, MS2 phage has been especially popular
for packaging of RNA. Encapsulation of a cocktail of anti-cyclin
small interfering RNAs (siRNAs) to silence expression of cyclin
A2, cyclin B1, cyclin D1, and cyclin E1 in MS2 was shown to
protect the RNA from degradation for over 3 months when
stored in the refrigerator.277 Targeting these VLPs to hepato-
cellular carcinoma cells resulted induced apoptosis in over 90%
of the cells at 150 pM siRNA concentration, with no substantial
effect on the viability of normal hepatocytes. siRNA delivery
with MS2 has been corroborated, with delivery of Bcl2 siRNA
packaged within MS2 targeted to TfR of HeLa cells causing
enhanced gene knockdown and apoptosis compared to non-
targeted particles and having effectiveness similar to a commercial
lipid transfection reagent.20 miRNA-mediated RNA interference
(RNAi) has also been explored, with MS2 conjugated with HIV-1
Tat47–57 peptides shown to be able to effectively transfer encapsu-
lated pre-miRNA into a range of cell lines and tissue types, which
was processed into mature miRNA and subsequently suppressed
expression of specific target gene.278 The same group also showed
packaging of antisense RNA delivered with the same Tat peptide
for inhibition of hepatitis C virus (HCV) RNA translation.279 While
so far these studies have mainly been proof-of-concept, they
suggest VLPs have much potential as gene delivery systems, with
many directions for gene therapy applications.
4.1.5 Drug delivery. In addition to gene delivery, viruses
can also be used for the delivery of drugs, where viruses are
applied as carriers of therapeutic cargo for photothermal
therapy, photodynamic therapy (PDT), and chemotherapy. Photo-
thermal therapy is an area whose potential has barely been
tapped in the virus realm. In general, heat is produced by metallic
nanostructures through the absorption of NIR or infrared light to
induce hyperthermia, which can be utilized for killing susceptible
tumor cells. Using such an approach, treatment can be applied to
a specific area and toxicity elsewhere is reduced. So far, there has
been one study, in which 1.3 nm gold was covalently attached to
adenovirus and delivered to HeLa cells, where the possibility of
using virus-based particles for a combination of photothermal
and gene cancer therapy was discussed.280 Other studies since
then have also been successful in attaching gold to VNPs,281,282
but photothermal therapy using these formulations has yet to be
explored.
Photodynamic therapy, like photothermal therapy, utilizes
light as a trigger, except with the effect of creating localized
cytotoxic reactive oxygen species (ROS) for therapy. Initially,
PDT was applied as an antimicrobial approach, using CCMV
functionalized with a ruthenium-based photosensitizer and
directed to Staphylococcus aureus using both an electrostatic
approach with PLL and a targeted approach with an antibody
for protein A, which is present in the cell wall.283 PDT was
demonstrated at standard antimicrobial fluence rates of up to
55.2 J cm2, with cell reduction of about 3 orders of magnitude
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Fig. 11 Gene delivery to mammalian cells using CCMV plant virus. (a) Strategy for delivering DI[EYFP], defective interfering RNA for enhanced yellow
fluorescent protein (EYFP) derived from SINV, through cotransfection of CCMV containing the gene with Lipofectamine-2000. (b) Flow cytometry
analysis of transduction efficiency showing lower efficiency for VLP transduction than naked RNA but the cargo is protected from RNase A.
(c) Corresponding fluorescence microscopy images (columns are in same order as bar graph) showing EYFP signal due to transduction for all conditions
except for naked RNA incubated with RNase A. Reproduced with permission from ref. 276. Copyright 2013 Elsevier.

observed and more selectivity imparted through the use of anti-
protein A for targeting. Since then, PDT has been applied as a
therapeutic against cancer cells. The buckyball C60 has been
attached to CPMV and Qb to improve solubilization of the
photosensitizer,94,284 and to aid in delivery to and treatment of
PC-3 prostate cancer cells.284 Internal conjugation of porphyrins
to MS2 combined with external display of aptamers for protein
tyrosine kinase 7 receptors on Jurkat leukemia T cells has been
established for selective killing of Jurkat cells when cultured with
erythrocytes after 20 min of illumination at 415 nm.285 Addi-
tionally, as a proof-of-concept study, glycan decoration of Qb was
shown to be an effective strategy for targeting cells bearing CD22
receptors that are involved in the regulation of B cells, and this
was illustrated with the delivery of metalloporphyrins for specific
elimination of CD22-positive cells.286 To move toward clinical
application of PDT, use of photosensitizers that can be excited in
the NIR range would be more ideal for better tissue penetration.
Phthalocyanine dyes are one such class of photosensitizers,
and self-assembly of CCMV with phthalocyanine encapsulated
has been explored recently.125,287 CCMV coencapsulated with
Gd(DOTA) micelles and phthalocyanine dyes resulted in better
capsid stability and further imparts the possibility to perform
combination therapy and MR imaging,287 and thus it only
remains to investigate their potential value in PDT.
Virus-based and other nanoparticles have also been developed
for the delivery of chemotherapies. Chemotherapy is associated
with dose-limiting toxicities, and specific delivery to cancer cells
using carrier systems increases safety as well as targeted payload
delivery.288,289 In particular, doxorubicin (Dox) delivery has been
studied extensively, likely popular in part due to the clinical
success of Doxil, a liposomal formulation of Dox.290 Dox works
through intercalating into DNA and causing oxidative DNA
damage.291 Conjugation of Dox can be achieved by pH-cleavable
hydrazone linkages, as demonstrated in one study where an
alkyne-functionalized hydrazone linker was used for attachment
to azide-functionalized polymers grown from the capsid of Qb by
ATRP.105 In such a manner, the Dox could then be subsequently
released by low pH cleavage of the linker after particle uptake,
which was shown to maintain efficacy in the killing of HeLa cells.
Methods for conjugation have also been recently established for
spheres made from thermal transitioning of TMV and utilized
for the loading of doxorubicin, which was found to be effective for

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chemotherapeutic delivery to breast cancer cells.292 Besides
chemical conjugation, Dox can be loaded by infusion into
RCNMV and incorporated into a fibrous matrix made up of
polylactic acid (PLA) and polyethylene oxide (PEO) nanofibers.113
The combination of the two systems could be tailored to result in
either a two-phase release profile or a first order release profile,
depending on whether the virus was co-spun with the fibers or
the matrix immersed in the virus solution after electrospinning,
respectively.
Specific delivery of Dox to cells has been achieved through
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of OVCAR-3 ovarian cancer cell growth was achieved, while no
difference in drug efficacy was observed when CCL-186 human
diploid fibroblast cells were tested as a control for normal cells
(Fig. 12).184 As another example, CPMV was used for treatment of
HeLa cells through the display of Dox attached either by a direct
covalent bond or through a disulfide linker.295 With the disulfide
linkage, the cell killing was similar to free Dox, likely due to
release of the drug in cell culture media before particle uptake,
but covalent attachment of Dox to CPMV resulted in more
efficient cell killing than free Dox, with almost complete elimina-

conjugation of FA to HCRSV and CMV,163,184 SPARC binding
peptides to M13,293 and peptides for CD46 receptor and
N-cadherin targeting to RCNMV,294 as well as by utilizing CPMV’s
natural interactions with surface vimentin.295 For instance, with
Dox encapsulated within Hibiscus chlorotic ringspot virus
(HCRSV) further conjugated with FA, more efficient inhibition
tion of the cells at a concentration of 1.45 mM for the CPMV
formulation whereas cells treated with free Dox were still
completely viable.
Virus-based platforms have also been explored for the
co-delivery of therapies. In one study, M13 was investigated
for its feasibility in delivering hygromycin and Dox specifically

Fig. 12 FA targeting for specific cell killing with Dox. (a) Schematic of formation of HCRSV-based protein cages without (PC-Dox) and with FA
conjugation (fPC-Dox) where Dox is encapsulated during capsid reassembly with the inclusion of polyacid. (b) Confocal microscopy of Dox uptake for
OVCAR-3 ovarian cancer cells and CCL-186 fibroblast cells incubated with free doxorubicin, PC-Dox, fPC-Dox, and fPC-Dox in the presence of FA.
(c) Cell viability curves of cells after treatment with varying concentrations of the different formulations showing fPC-Dox had greater inhibition of
OVCAR-3 cells without affecting pattern of CCL-186 inhibition. Reproduced with permission from ref. 184. Copyright 2007 American Chemical Society.

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to either SKBR3 human breast adenocarcinoma cells that over-
express HER2 or A431 human epidermoid carcinoma cells that
overexpress EGFR.296 Dox delivery was successful and was
shown to require a cathepsin-B cleavable peptide linker for
efficacy, but delivery of hygromycin was particularly impressive,
with over a 1000-fold improvement in potency compared to free
drug administration. However, different targeting strategies
were used, making it difficult to draw conclusions as to the
relative efficacy of the two drugs. Another study assessed the
range of therapeutic cargo that could be encapsulated within
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MS2, including doxorubicin, cisplatin, and 5-fluorouracil
(5-FU), along with ricin toxin A-chain (RTA).277 Using SP94-
targeted formulations that bind to Hep3B hepatocellular carcinoma
(HCC) cells with 104-fold higher avidity compared to other cell
types, the encapsulation of Dox individually was shown to be more
effective than free Dox, while encapsulation of a cocktail comprised
of all three chemotherapeutics was even more effective, with an IC50
concentration below 1 nM. Delivery of the toxin RTA was also
remarkable, with almost complete elimination of Hep3B cells
at a concentration of 100 fM without affecting the viability of
control cells.
While the targeted chemotherapeutic cocktail above was
highly effective, other approaches using prodrugs have also
been investigated to further reduce the risk of toxicity. For
example, instead of direct delivery of 5-FU as a treatment
option, encapsulation of the enzyme yeast cytosine deaminase
(yCD) is an alternative method that results in the presence of
5-FU in the target cell by conversion of 5-fluorocytosine and as a
consequence causes target cell death.297 In another study that
utilized conversion of a prodrug, delivery of an exogenous
protein horseradish peroxidase (HRP) was demonstrated with
M13 displaying Ypep2 peptides, which have selectivity for PC-3
prostate cancer cells.298 After delivery, HRP was able to oxidize
indole-3-acetic acid to produce a peroxyl radical that led to
cytotoxicity.
Other anticancer drugs that have been investigated for virus-
based nanoparticle delivery include taxol,299 bortezomib (or
BTZ),300 and trastuzumab (or Herceptin).144 Cardiovascular
disease is another route for virus-based therapeutic inter-
vention, and CPMV delivery of chromium has shown promise
for protecting against diabetic atherosclerosis in vascular smooth
muscle cells.301 Additionally, filamentous phages have been
applied for the delivery of antibacterial agents, including
neomycin and chloramphenicol for growth inhibition of
E. coli, S. aureus, and Streptococcus pyogenes.302,303 Antiviral
delivery with viral vectors is a new development, and the similar
tropism of the plant virus CPMV to antigen presenting cells that
are commonly subverted by pathogenic viruses was exploited
for combatting chronic infectious disease caused by the prototypic
arenavirus lymphocytic choriomeningitis virus.111
Only very recently was drug delivery with VNPs or VLPs
demonstrated in vivo.304 Since TMV is a hollow nanotube with
a negatively charged interior channel, it could be taken advantage
of for the loading of cationic drugs. The highly potent platinum
DNA-binding drug phenanthriplatin was introduced within the
carrier and shown to be released under acidic conditions.
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When TMV delivery of phenanthriplatin was applied in a triple
negative breast cancer mouse xenograft model, much greater
efficacy was observed for TMV-phenanthriplatin compared to
free drug or clinically used cisplatin controls, with 4-fold
smaller tumor growth. This is likely due to better transport of
the drug to the tumor cells with the nanocarrier. The combi-
nation of TMV and phenanthriplatin shows potential for bring-
ing an effective new chemotherapy into the clinic, but virus-
based nanoparticle drug delivery is a quickly growing field, and
it is expected that progression toward other clinical applications is
also fast approaching.
4.1.6 Tissue engineering. Viruses have been incorporated
into biocompatible tissue engineering scaffolds for directing
cell growth, alignment, and differentiation. The neighboring
environment affects the behavior of cells, both in terms of
biological cues such as presence of chemokines and ligands as
well as physical cues such as topology and mechanical moduli.
Using this knowledge, scaffolds can be designed to regulate
cells in a manner suitable for applications in the repair or
replacement of damaged tissue.
As a first step, cell adhesion to viral scaffolds was investi-
gated. Pioneering work utilized layer-by-layer (LbL) assembly of
CPMV and polymer poly(diallyldimethylammonium chloride)
(PDDA) to form thin films that were found to aid in the
adhesion and proliferation of NIH-3T3 fibroblasts, with more
layers leading to more CPMV adsorption and greater cell
adhesion.305 Research then moved toward the use of filamentous
viruses, as they better mimic the structure of the extracellular
matrix (ECM). Coating of TMV with different cell binding motifs
derived from integrin binding matrix proteins collagen and
fibronectin onto a high binding plate demonstrated that the
peptide sequence displayed plays a role in cell adhesion and
morphology.306 While cells cultured on TMV with RGD motifs
formed filopodial extensions, they adhered more weakly compared
to cells that remained rounded when cultured on TMV displaying
P15, a collagen I mimetic sequence. For screening the influence of
various biochemical cues on cell proliferation and morphology,
phage-chips have been constructed such that arrays of M13
nanofibers labeled with various peptides or growth factors and
self-assembled on gold chips can be monitored using surface
plasmon resonance (SPR) spectroscopy for their effect on cultured
cells.307 Further progression toward mimicking the ECM looked
at synthesizing fibrous matrices through electrospinning of RGD-
modified viruses with polymers.308,309 Nanofibers made up of TMV
with polyvinyl alcohol (PVA)308 and M13 with poly(lactic-co-glycolic
acid) (PLGA)309 formed biodegradable fibrous matrices that
enhanced cell adhesion, proliferation, and spreading of baby
hamster kidney (BHK) and fibroblast cells, respectively, compared
to scaffolds of the polymers alone.
As an advancement from cell adhesion and proliferation,
external stimulus can also direct the orientation of cell growth,
which is important for the function of many cell types such as
cardiac and skeletal myocytes. Taking advantage of available
viruses with elongated geometries, M13 and TMV have been
utilized for cell alignment. In an early study, M13 with RGD
motifs was self-assembled into parallel arrangements through
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slow drying or dragging methods to form thin films on which
oriented growth of NIH-3T3 and Chinese hamster ovary (CHO)
cells were achieved, with the shear method producing the most
consistent results.310 Investigation of the ECM deposited by
cells cultured on these films revealed correlation between
cellular alignment and the orientation of fibronectin and
collagen I deposition.311 NIH-3T3 fibroblasts produced more
ECM proteins, which resulted in more of a tendency to deviate
from the original patterning, and thus the fibroblasts displayed
reduced alignment over time as well as when compared with
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less ECM-producing BHK cells. Other methods for the alignment of
viral nanorods, and subsequently cells, include shearing force from
fluid flow through glass capillaries312,313 and microcontact printing
combined with dip-coating.314 Myogenic differentiation of myoblast
cells can be achieved through exposure to differentiation media
after oriented cell growth.313
In addition to forming 2D films, fabrication of 3D aligned
fibers is possible based on interfacial polyionic complexation,
which was demonstrated by injecting negatively charged, RGD-
labeled M13 phage into a solution of cationic polymers PEI,
PLL, and chitosan.315 NIH-3T3 fibroblast cells encapsulated
through co-injection with the phage solution grew well within
the fibers and after seven days began to spread along the matrix
within the phage fibers, demonstrating the potential of virus
scaffolds for cell growth and remodeling. 3D tissue cultures
have also been formed through treatment of cells with hydrogels
that can then be magnetically levitated, and cell clustering could
be controlled by the magnetic field profiles of the magnets
used.316 Hydrogels were fabricated through simple combination
of solutions of gold nanoparticles with M13 displaying
RGD,317,318 with the additional inclusion of magnetic iron oxide
nanoparticles for levitation. The 3D cell cultures were able to
recapitulate in vivo behavior, as observed by similar protein
expression patterns as well as infiltration of highly invasive
glioblastoma cells when co-cultured with astrocytes.
An application of phage nanofiber formation is the growth
and differentiation of neural cells. Fibers containing M13
displaying RGD or IKVAV, a laminin motif that plays a role in
neural cell adhesion and neurite extension, were shown to be
advantageous for neural progenitor cell (NPC) proliferation and
differentiation, with extension of neurites parallel the fibers
observed.82 The cell binding motifs are crucial for inducing
neural cell growth, and it has been demonstrated that using a
control RGE peptide results in a drastic decrease in cell adhesion
and neurite outgrowth.319 There is a range of cell binding
peptides derived from fibronectin and collagen that have been
investigated and found to be effective for supporting neural
growth and enhancing neurite extension,320 and it would be of
interest to further study the specific interactions between the
cells and the scaffold to better understand the roles of the
peptides in cell differentiation. In addition to cell binding motifs,
immobilization of growth factors to phages has been demon-
strated to retain bioactivity, with bFGF shown to promote NPC
proliferation and NGF leading to greater neural differentiation.321
Furthermore, a recent study demonstrated that electroactivity
could also be used to augment neural tissue regeneration.322
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TMV that was modified with polyaniline and further doped
with poly(styrenesulfonate) (PSS) was aligned by flow through
capillary tubes and shown to increase the percentage of cells
with neurite outgrowths as well as the percentage of cells with
bipolar morphology.
Virus scaffolds are not limited to neural differentiation, and
by far the most extensive research has been performed in
osteogenic differentiation. An early study looked at osteoblast
differentiation of bone marrow stromal cells on icosahedral
turnip yellow mosaic virus (TYMV).323 Osteocalcin gene expres-
sion and onset of mineralization were found 7 days earlier for
cells grown on TYMV-coated substrates compared to tissue
culture plastic when cultured in osteogenic media, indicating
the nanotopology imparted by the TYMV supports osteogenic
differentiation. Similar data was found for substrates coated
with rod-shaped TMV, with further DNA microarray data showing
differential expression in a large panel of genes and bone
morphogenetic protein 2 (BMP2) in particular found to be
especially important in osteogenic differentiation in this
manner.324 There was a rapid onset in BMP2 gene and protein
expression, and this enhancement was only found when TMV
was coated on the substrate and not when added to the cell
media, verifying the role of nanotopology.325 Additional con-
jugation of phosphate groups to the exterior of TMV further
boosted differentiation by aiding in the incorporation of calcium
and consequently highly enriched mineralization of the ECM.326
A related strategy for mineralization utilized genetic engineering
to display highly negatively charged E8 peptides on M13 phage,
which could then be self-assembled into nanofibers in the
presence of calcium ions and led to hydroxyapatite formation
with the addition of phosphate ions.327
The effect of multivalent presentation of various ligands has
also been studied, and rapid differentiation and bone-like
nodule formation was observed for substrates with TMV coated
with RGD after only 2 days in serum-free osteogenic media.328
While this was found for TMV genetically engineered to display
RGD, TMV chemically conjugated to RGD also enhanced bone
differentiation.329 In the absence of osteogenic supplements,
presentation of RGD with the addition of synergy peptide
PHSRN on M13 was actually found to be sufficient to induce
osteoblastic differentiation of mesenchymal stem cells (MSCs).330
Display of DGEA peptide derived from collagen331 along with
PDPLEPRREVCE derived from osteocalcin and YGFGG derived
from osteogenic growth peptide332 on M13 have also been shown
to accelerate proliferation and differentiation of MSCs into osteo-
blasts. M13 is capable of self-assembly into long-range ordered
morphologies using dip-coating methods, forming nematic
orthogonal twists, cholesteric helical ribbons, and smectic
helicoidal nanofilaments based on conditions such as phage
concentration and pulling speed.333 Films formed using RGD
and EEEE peptide-labeled phages could be used to control both
soft and hard tissue formation, with smectic hilcoidal nano-
filament surfaces in particular demonstrated to form enamel-
like composites when treated with calcium and phosphate ions.
Moving toward 3D scaffolds, TMV-RGD was incorporated within
porous alginate hydrogels and resultantly produced greater cell
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attachment and enhanced osteogenic differentiation.83 3D
printed scaffolds composed of hydroxyapatite and b-tricalcium
phosphate is another approach, and introduction of M13-RGD
combined with chitosan within the scaffold pore not only led to
osteogenesis but also angiogenesis, with inclusion of VEGF
further enhancing the effect (Fig. 13).334
A new direction in this area is the incorporation of gene
delivery within tissue engineering constructs. Mutant FLAG-
tagged AAV was tethered to scaffolds made up of PLGA and
gelatin sponge through anti-FLAG antibodies, and virus trans-
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cassette.336 While still in its early stages, with further developments,
tissue regeneration and reprogramming of cellular defects could be
made possible through the combination of tissue engineering and
gene delivery.
For future translation of virus-based tissue scaffolds, the
immunogenicity and long-term effects of the viruses must be
considered. Investigation of the in vivo behavior of implanted
porous alginate hydrogels containing TMV and TMV-RGD
revealed good biocompatibility, as evidenced by normal wound
healing, hydrogel biodegradation over time, no pathological

duction was observed when HeLa cells were seeded onto the
scaffold, both in vitro and when implanted in vivo into nude
mice.335 Furthermore, transduction was observed for cells cultured
on drop-cast films consisting of hybrid phages constructed from
the combination of M13-RGD phage with an AAV-derived gene
inflammation, and very little immune response triggered as
opposed to intramuscular injection of native TMV.337 In addi-
tion to degradation of the hydrogel, it is expected that the
protein-based viruses will also be degraded over time by cell
proteinases, thus mitigating concerns of the implications of

Fig. 13 3D printed virus-activated bone scaffold with angiogenesis. (a) Schematic of 3D printed bioceramic bone scaffold incorporating negatively
charged RGD-labeled phage nanofibers using positively charged chitosan for new bone and blood formation when seeded with MSCs. (b) Images of
scaffold architecture. Scanning electron microscopy (SEM) of bone scaffold showed macro-scale (1) and micro-scale (2) pores, as well as pores filled with
chitosan and phage matrix (5). Atomic force microscopy (AFM) (3) and transmission electron microscopy (TEM) (4) demonstrated morphology of phage
nanofibers. 3D confocal fluorescence imaging showed presence of dye-labeled phage (red) within matrix-filled pores (6), and brightfield imaging
revealed support of MSC adhesion for both the scaffold pores (7) and columns (8). (c) Immunofluorescence staining for endothelial CD31 (1, 3, 5) and
hematoxylin and eosin (H&E) staining (2, 4, 6) of implants of negative control (wild-type phage), virus-activated matrix (VAM), and positive control
(RGD-phage with VEGF) scaffolds, respectively, as well as quantitative analysis (7) showed VAM promotes angiogenesis at an intermediate level.
(**p o 0.01). Reproduced with permission from ref. 334. Copyright 2014 John Wiley & Sons.

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long-term deposition of the material. Since previous work has
shown that viral substrates can be utilized for rapid differentiation
in serum-free media, thus without the presence of xenogeneic
proteins and growth factors,328 implantation of tissue scaffolds
after ex vivo culturing of cells is a tangible reality.
4.2 Biotechnology
In the realm of biotechnology, viruses have found use for a
variety of applications including peptide display technologies,
confined synthesis, multiplexed sensors, diagnostics, nano-
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reactors, catalysts, as well as agriculture, several examples of
which are discussed in the following sections.
4.2.1 Phage display technologies. The first of these techno-
logies that we will cover is phage display, which is routinely
applied for a myriad of applications. The display of foreign
sequences on filamentous phages was first described by George
Smith in 1985.338 Since then, it has become a prominent
method for the selection of peptides and antibodies that have
affinity for specific targets, encompassing both organic and
inorganic matter. Many reviews have been written that delve
into the depths of phage display technology.339–343 Our aim in
this section is to discuss the characteristics of phage display
and highlight a few of the interesting studies and some of the
latest applications.
Based on the original research in which f1 phage was
studied,338 E. coli filamentous bacteriophages, which include
f1, fd, and M13, are some of the more common platforms
utilized for phage display. The general structure of filamentous
phages is shown in Fig. 14,344 which illustrates how the phages
are comprised of a number of minor and major coat proteins.
Out of these, the pIII and pVIII proteins are what are usually
utilized for the display of foreign proteins,345 likely due to
greater accessibility at the tip and sides, respectively, but display
with pVI,346 pVII,347,348 and pIX348 have also been successfully
implemented. In brief, phage display utilizes the genetic
programming of the phage coat proteins; insertion of random
Fig. 14 Filamentous phage structure. (a) Schematic of phage structure
showing how the five structural proteins are arranged around its ssDNA
genome. (b) Legend labeling the structural proteins with approximate
values for size, weight, and copies/virion. Reproduced with permission
from ref. 344. Copyright 2011 Løset et al.
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sequences of DNA within the protein genes can be used to form
a library of phages with billions of different foreign peptides or
proteins presented.342 After several rounds of panning and
amplification to isolate the specific phages that bind to a target,
the identity of the binding peptides or proteins displayed can
be determined through sequencing. Whereas insertions at
pVIII are limited to around 9-mers,349 cyclic phage libraries350
and libraries displaying sequences of up to 38 random residues
on pIII are possible.351 Phage display is not only limited to
filamentous phages, as libraries based on other phages such as
lambda and T7 are also possible.352,353
Another alternative that has proven useful in phage display
is the use of phagemids.354 Phagemids typically contain tradi-
tional plasmid aspects, with a plasmid replication origin,
restriction enzyme recognition sites, and an antibiotic selection
marker, as well as phage aspects, including a phage origin of
replication and a gene for pIII fused to any protein of interest
(Fig. 15).355 By co-infection with a helper phage to supply other
structural proteins required for phage formation, complete
virions assembled around phagemid DNA can be recovered.
The advantages of using phagemids include ease of cloning and
recombination, more efficient transformation, a smaller genome
for incorporation of larger foreign genes, and greater genetic
stability over multiple rounds of propagation.354
Phage and phagemid display technology have given rise to
numerous opportunities for the isolation of protein-based
ligands for a range of applications. In its simplest form, screening
can be performed in vitro, where the target of interest is immo-
bilized on a solid support. Using this technique, peptides have
been identified that are specific for targets that include inorganic
materials such as hydroxyapatite,356 silver,357 and quantum
dots,13 as well as organic materials such as microtubules,358
fibrin,359 and integrins.360 As some examples, these binding
peptides can be used for biomineralization for formation of
hard tissue356 as well as for nucleation, growth, and patterning
of metals.357 It should be noted that many of the examples
found in other sections that involve mineralization rely on the
use of metal binding peptides for nucleation. Additionally,
peptides can be selected that not only bind but also obstruct
the function of its targets, such as demonstrated with peptides
identified for inhibition of proteases such as human neutrophil
elastase,361 as well as cancer-associated matrix metalloproteinases.362
The versatility of the technology can be expanded beyond
peptide identification. For example, phage libraries can be
designed for the display Fab antigen-binding fragments and
scFvs,339 which has implications for identification of anti-
bodies for bioassays as well as immunotherapy. Some examples
include the construction of a Fab phage display library for the
isolation of antibodies specific to human prostate cancer
cells363 and the generation and identification of scFv anti-
bodies that recognize a marker of angiogenesis, VEGFR-3.364
Besides Fabs and scFvs, an alternative method of display where
the antibody heavy- and light-chain variable regions were
separately displayed on pVII and pIX, respectively, was demon-
strated to effectively drive the formation of functional Fv hetero-
dimers, which may have greater affinity and stability compared
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Fig. 15 Phage display cycle with phagemid. A library of DNA sequences with random variations of the protein of interest (POI) displayed on the pIII coat
protein is cloned into a phagemid vector. After transformation of E. coli cells and subsequent infection with helper phages, the phage library is created.
Using an immobilized target molecule, rounds of selection and amplification are performed until phages with the highest affinity are isolated. DNA
sequencing can be used to identify the phages, and/or directed evolution can be used to create new libraries for panning. Reproduced with permission
from ref. 355. Copyright 2011 Biochemical Society.

to scFvs.365 Enzymes and their substrates can also be displayed
on phages, either independently or together on a single particle,
in order to screen for functional enzyme catalysts.366
Co-presentation of the enzyme and substrate can be achieved
using an intervening linker between the two, and then active
enzymes can be selected for by panning for product formation.367
Aside from immobilization of isolated targets on a surface,
selection methods have also been demonstrated with cultured
cells, as well as ex vivo and in vivo, to select for cell binding
peptides.343 Using panning against cell lines of various types,
such as fibroblasts and myoblasts,368 peptides with high
cellular specificity can be identified. In this manner, peptides
have been isolated that have specificity for endothelial cells
associated with atherosclerosis,369 breast cancer cells,370 hepato-
cellular carcinoma cells,371 melanoma cells,372 and ovarian
cells,373 among others. Ex vivo panning works similarly, except
the library is screened against cells or tumor masses that have
been isolated, and binding peptides have been identified for
a range of cell types that include neuroblastoma cells,374 islet
cells,375 and colonic adenoma cells.376
In vivo phage display is particularly noteworthy and lends
itself to many different applications. For example, peptides
targeting specific organs can be examined using in vivo screening
of random peptides in a mouse model.377 After multiple rounds of
phage administration, isolation, and amplification, peptides that
localize to the brain and kidney were identified. Furthermore,
attachment of brain targeting peptides to fixed red blood cells
resulted in the accumulation of the cells in the brain at a greater
extent than the kidneys. Another investigation that also studied
kidney targeting in vivo found that peptide specificity could be
used for the tailoring of pharmacokinetics.378 By directing
clearance toward the kidneys and away from the reticulo-
endothelial system, more rapid clearance could be achieved.
In vivo screening has also been applied in humans for the
mapping of unique zip codes lining the vasculature.379 Unique
tripeptide motifs specific for various regions around the body
were identified after biopsies of the bone marrow, fat tissue,
skeletal muscle, prostate, skin, and liver were performed, which
could be utilized toward the creation of a map of molecular
signatures along the human vasculature.
As the above examples indicate, the reach of phage display
applications is extensive and encompasses mineralization,
in vitro assays, targeted delivery, molecular imaging, vaccines,
and tissue engineering.
4.2.2 Sensing and multiplexed systems. The multivalency
of viruses can be applied for sensing and multiplexed systems;
the high degree of multivalency has been shown to improve the
detection limit in a number of settings. Phages identified from
a library against the desired target sequence can be directly applied
as sensors. Genetic engineering simplifies the manufacture of large

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quantities of phages displaying specific targeting moieties, and
several reviews cover the wide range of sensing applications
available for these materials.380,381 Besides phages, many other
virus-based platform technologies have also been developed for
signal detection and amplification. In our examples, we will cover
the broad range of virus-based materials incorporated in manifold
sensing technologies, which include antibody-based, electro-
chemical, and optical techniques.
Enzyme-linked immunosorbent assay (ELISA) is a traditional
technique for the detection of target antigens.382 Although several
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variations exist, it generally relies on three components: (1) a
probe specific for the target, (2) an antibody tagged with an
enzyme to detect the probe, and (3) a substrate that is converted
in the presence of the enzyme. The first probe tends to also be an
antibody with specificity for the target, but phage display has
introduced the opportunity for using peptides for target recogni-
tion. For example, phages decorated with peptides isolated
from phage display have been used in ELISAs for the detection
of anthrax spores383 and the surface antigen of HBV.384 For an
alternative to the presentation of peptides on viruses, antibodies
can also be presented on the viral scaffold for multivalent
detection, and the previous section discussed how phage display
and genetic engineering could be used for the display of
antibodies and fragments thereof. As another approach, it was
recently demonstrated that functionalization of PVX-based nano-
particles with protein A fragments can be used to display whole
antibody molecules by using protein A’s property of binding to
the Fc region of the heavy chain of IgGs.85 The PVX particles
could then be used as a plug-and-play system for the display of a
variety of antibodies, with a level of orientational control not
achievable with chemical conjugation.
Instead of sample immobilization followed by probing for
antigens in the sample with a virus-based detector, viruses
displaying antigens could first be immobilized before incubation
with the sample to detect for the presence of certain anti-
bodies.385 This was demonstrated as a successful approach for
the diagnosis of primary Sjögren’s syndrome (pSjS), a chronic
systemic autoimmune disease whose heterogeneity often delays
diagnosis. Lipo peptide derived from the human autoantigen
lipocalin was displayed on PVX, and the nanoparticle platform
was shown to be specific to pSjS patient sera and had greater
reactivity than the peptide alone. On the other hand, eCPMV-
based display of lipo was not reactive, likely due to a smaller
density of peptide display. Regardless, the PVX ELISA has promise
for future implementation, and an additional benefit found was
the stability of the coating, with no loss of specificity or sensitivity
observed even after two months of storage at 4 1C.
Another aspect of ELISAs is the enzyme used for quantification
via substrate conversion. HRP is a popular enzyme for ELISAs
due to its ability to convert chromogenic substrates into colored
products and chemiluminescent substrates into fluorescent
products.382 Immobilization of HRP, as well as glucose oxidase
(GOX), has been explored using CPMV386 and TMV387 platforms.
While the addition of sensing molecules to the virions will be
required for their use in ELISAs, the display of the enzymes
themselves on the particles can be directly used for sensing.
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GOX catalyzes the oxidation of glucose and concurrently
generates hydrogen peroxide, which can then be reduced to
water by HRP in the presence of a substrate. Therefore, the two
enzymes can be coupled to form a glucose sensing system,
where HRP substrate conversion can be used for detection.
In fact, the TMV nanorods resulted in up to 45-fold higher
substrate conversion rates than control samples with the same
input of enzymes, which could be due to a combination of
a greater surface area and better steric accessibility of the
presented enzymes. This is only one example of the potential
of enzyme-based sensors, and the immobilization of enzymes
that catalyze other reactions could also be considered for the
creation of sensitive biosensors.
The output of immunosorbent assays need not be enzyme-
linked, and an alternative readout options include fluorescence
from the particles. For example, phages isolated after panning
against staphylococcal enterotoxin B (SEB), an agent that can
cause food poisoning, were labeled with the fluorophore Cy5
and used to probe for SEB.388 Based on fluorescence readings,
SEB was detectable down to a concentration of 1.4 ng per well.
As another example, dye-labeled CPMV conjugated with anti-
bodies was used for the detection of SEB, botulinum toxin, and
the bacterium Campylobacter jejuni.389 The detection of SEB was
specifically quantified, and the limit of detection was improved
for the CPMV formulation when compared with a mole equivalent
of dye-labeled antibody.
Fluorescence can be used for other sensing functions,
such as found with the application of fluorescently labeled
CPMV to DNA microarray sensors.390 By additionally coupling
NeutrAvidin to the capsid, the CPMV could be used as a
detection reagent. One result of the investigation demonstrated
that the delivery of multiple dyes using CPMV resulted in signal
amplification and led to the detection of 14% more genes
compared to the control in a rat expression array (Fig. 16).
Another fluorescence sensing application utilized LbL assembly
of M13 labeled with quantum dots along with quenchers that
can be displayed by the explosive 2,4,6-trinitrotoluene (TNT).391
The design of the M13 film allowed for highly selective detection
Fig. 16 Microarrays hybridized with cDNA made from rat total RNA.
(a) Result from cDNA labeled with Cy5-dCTP control. (b) Result from
cDNA labeled with biotin-functionalized dCTP and dUTP followed by
binding with NeutrAvidin-functionalized CPMV-Cy5. Both strategies
resulted in a density of labeling of about one every 50 bases, but the
CPMV-based probe resulted in greater sensitivity, detecting 71% of the
features compared to 57% for the control. Reproduced with permission
from ref. 390. Copyright 2009 Elsevier.
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of TNT at a sub ppb level through the evolution of fluorescence
signal when the quenchers were displaced.
Optical sensors also encompass those that utilize SPR for
detection. For example, LbL assembly of cationic M13 with
anionic gold nanoparticles resulted in the development of an
SPR spectrum that was sensitive to humidity.392 It is therefore
possible to utilize electrostatic assembly to integrate viruses in
humidity sensing devices. SPR has also been applied as sensors
for immunoassays, such as exemplified by the detection of the
food-borne bacterium Listeria monocytogenes using a gold SPR
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sensor chip.393 M13 displaying an scFv antibody recognizing
L. monocytogenes cells was immobilized on the sensor chip
before injection of samples for measurement. The change in
resonance due to the binding of cells allowed for specific
detection down to levels of around 2  106 CFU per mL.
Surface enhanced Raman spectroscopy (SERS) has been another
approach that has been considered for sensing.394 It relies on signal
enhancement of the Raman signal when in close proximity to the
surface of a noble metal. Due to the sensitivity of the technique and
the unique signature of various reporters, SERS is a promising
technique for multiplexed analysis. M13 phage, selected by phage
display for binding to the model rabbit anti-goat IgG antigen, was
labeled with Cy3 Raman reporters that were conjugated to Au@Ag
core–shell nanoparticles. Due to a high surface area for reporter
presentation, the resultant M13 construct imparted an exponential
increase in the Raman intensity observed when compared to
similarly labeled antibodies against the model antigen. M13 has
also been applied in a colorimetric sensor that was based on
changes in the modulation of its self-assembled structure, but this
application is discussed in more detail in Section 4.3.1.395
Besides optical sensing, viruses have also found application
in electrochemical sensing. The three different examples
we will highlight involve electrochemical impedance spectro-
scopy396 and amperometry397,398 for measurements. In the first
example, a gold electrode surface on which a M13 monolayer
was attached was used for the detection of an antibody as
well as prostate-specific membrane antigen (PSMA), a marker of
prostate cancer.396 The resistive component of the impedance,
ZRe, measured from 2 to 500 kHz, increased upon analyte binding,
and measurement of this characteristic could be used for highly
specific detection of down to concentrations of around 100 nM.
In the next example, a thin film of TMV conjugated with electro-
active oligoaniline was used for the detection of volatile organic
compounds, specifically methanol and ethanol.397 The response
current was measured from the thin film sensor, and there was a
high response observed in the presence of ethanol and methanol
over what was observed for acetonitrile, isobutyl alcohol, tetra-
hydrofuran, toluene, and acetone. Moreover, the current mea-
surements exhibited reproducibility as well as a quick response
time for both absorption and desorption of the compounds.
Finally, the last example involves the application of a solution
of TMV displaying binding peptides for TNT for in-solution
sensing of the molecule.398 As a result of binding of TNT to
TMV, the diffusion coefficient of the TNT was reduced and a
differential Faradaic current signature of the electroactive
compound was caused. The differential current was proportional
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to TNT concentration and therefore could be used for TNT
sensing. Detection using this method is not limited to just TNT
and can be expanded to other relatively small electroactive species.
4.2.3 Diagnostic controls. Another sensing approach,
which is widely applied for disease diagnostics, involves using
assays that detect and amplify the nucleic acid content of
infectious agents such as bacteria and viruses.399–401 Quality
control is an important consideration to ensure results from
these assays are accurate. For example, failures with the well-
known polymerase chain reaction (PCR) assay could occur due
to nucleic acid degradation or the presence of inhibitors such
as bile salts and polysaccharides in clinical samples, which
could potentially lead to reduction in polymerase binding or
activity.402 False negatives due to failures during processing
may be monitored by incorporating known RNA or DNA into
samples to serve as a positive internal control to verify that
nucleic acid degradation did not occur during processing.
To this end, viral capsids have found utility due to their
ability to shield nucleic acids from nuclease digestion, serving
as a better mimic for use in viral assays and allowing for
long-term storage of the controls. The design of so-called
‘‘Armored RNA’’ (developed and patented by Asuragen and
Cenetron Diagnostics) was the first such development and
functions as a well-characterized control that is resistant to
RNase.403 Armored RNA is comprised of an RNA standard
sequence encapsulated within an MS2 capsid, which can be
co-produced using an expression vector in E. coli (Fig. 17). If so
desired, the RNA within these particles can be subsequently
released through heating at 70 1C for 5 minutes. In the
pioneering study, the control was tested in an HIV reverse
transcription-PCR (RT-PCR) assay using a non-infectious con-
sensus sequence taken from the HIV-1 gag gene. The Armored
RNA particles were found to be stable in anticoagulated plasma
with no loss in signal after storage in a variety of conditions:
4 1C for 2 months, 20 1C for 6 months, and even after five
freeze-thaw cycles.
Fig. 17 Example of Armored RNA packaging. Transcribed recombinant
RNA, in this case an exogenous HCV-2b consensus sequence, can be
packaged within self-assembled MS2 coat proteins. Reproduced with
permission from ref. 404. Copyright 2009 American Association for
Clinical Chemistry.
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Since these initial results, Armored RNA has been applied
as a control for a vast array of assays, which include the
detection of HCV,404–406 respiratory viruses such as influenza
and severe acute respiratory syndrome (SARS) viruses,407–409
enteroviruses,410,411 and West Nile virus.412 It has even been
utilized as a surrogate virus for the detection of animal pathogens,
including classical swine fever virus (CSFV), FMDV, and vesicular
stomatitis virus (VSV).413 Several developments in the technology
have been made to enhance the utility of Armored RNA. In the
original area of HIV detection, Armored RNA was expanded
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beyond RT-PCR assays to the branched DNA (bDNA) assay, which
provides a reliable method for quantification of HIV-1 RNA but
requires a longer 3 kb RNA control.414 The standard strategy for
RNA encapsulation within MS2 is limited to only around 2 kb, but
increasing the packaging efficiency to overcome this limit can be
achieved through incorporating more translational repressor
stem-loops within the RNA, which specifically interact with the
MS2 capsid and trigger the self-assembly of the viral shell around
the cargo.415 In such a manner, Armored RNA containing the
longer HIV pol gene was successfully synthesized and per-
formed reliably as a standard for the bDNA assay.414 HCV
Armored RNA has also been developed for RT-PCR and bDNA
assays, and additional work demonstrated its applicability in
genotyping assays as well, allowing the distinction of a specific
subtype of HCV.404 For real-time RT-PCR reactions, false nega-
tives could occur when a singleplex primer/probe assay is used
since mismatches with a set of primers could exist in a number
of samples, thus dual-specific RNA controls in duplex assays
were tested, and resultant enhancement in the sensitivity of
detection was found compared to monospecific assays.405,416
Beyond controls for the detection of single virus types,
Armored RNA chimeras have also been created.407,417 In one
study, a chimeric RNA sequence was derived from a mix of gene
fragments from HCV, HIV-1, SARS coronavirus 1, and SARS
coronavirus 2.417 They were able to package the fairly large
1.2 kb RNA sequence within MS2 and demonstrate its versatility
as a control for the multiple different RT-PCR assays for the
detection of each individual virus. In another study, RNA
fragments from influenza A, influenza B, and SARS viruses
were spliced together into one fragment in order to create a
single control for the simultaneous testing of these common
respiratory viruses that may result in similar clinical symptoms.407
Using multiplex RT-PCR for the three viruses with different
reporter dyes, simultaneous amplification and detection could
be achieved with a highly sensitive detection limit of 101 copies
per mL of the Armored RNA for all the viruses.
A natural extension of Armored RNA technology is the
encapsulation of DNA within a bacteriophage capsid for quality
control of DNA viruses. Early work in this area utilized lambda
and filamentous fd phages for DNA packaging, and greater
stability of the DNA was observed.418–420 Containment of control
dsDNA within lambda phage conferred resistance to DNase
digestion, with reliable amplification in PCR assays for
HBV418,419 and cytomegalovirus.418 When stored in SM buffer,
the protected DNA was stable for at least a few months, but
stability for more than 5 days in plasma at room temperature
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could not be achieved, which may be a result of greater suscep-
tibility of lambda to plasma proteases compared to MS2.418,419
For ssDNA, encapsulation in fd phage was demonstrated with
DNA taken from parvovirus B19.420 The resultant controls were
resistant to nuclease degradation and performed similarly to
the native virus in PCR assays, with very similar growth curves
observed. Furthermore, the constructs remained stable at both
37 1C and 45 1C when diluted in human plasma for periods of at
least 4 weeks. More recently, a return to the Armored RNA roots
was taken with the demonstration of encapsulation of dsDNA
in MS2, which resulted in extra stability over lambda as was
found for fd phage but without being limited to ssDNA.421
By conjugating sulfhydryl-modified DNA sequences of interest
to an amine-modified stem-loop DNA structure, assembly of
dissociated MS2 CPs could then be triggered around the
stem-loops to form Armored DNA. Using this strategy, the
formation of MS2 capsids packaging HBV and HPV DNA
sequences with lengths ranging from 1.3 to 6.5 kb was accom-
plished, which is astonishing given that the genome of MS2 is
only 3.5 kb. The Armored DNA controls performed well in PCR
and genotyping assays, and storage in newborn calf serum even
after 6 months at 4 1C was shown to not affect performance.
Encapsulated controls are not only limited to bacteriophages.
For example, recombinant RNA particles based on CPMV can
also be utilized as internal controls for RT-PCR assays.422
To produce these particles, a cDNA clone was engineered to
contain sequences for the desired control RNA alongside RNA-2
of CPMV, which codes for its coat and movement proteins. In
the proof-of-concept study, two sequences from FMDV and one
from swine vesicular disease virus (SVDV) were cloned together
into the cassette. Using agroinfiltration of cowpea plants with
the combination of this plasmid along with another plasmid
for RNA-1 of CPMV, which is responsible for its replication
and proteolytic processing, recombinant CPMV particles were
propagated in and recovered from their host plant. The CPMV
component containing RNA-2 was separated from the RNA-1
component using a Nycodenz density gradient, resulting in a
non-infectious construct due to its reliance on RNA-1 for
replication. The particles performed well as positive controls
for the detection of both FMDV and SVDV. Additionally, they
were resistant to RNase and performed reliably even after
33 days storage in a 10% suspension of bovine epithelium at
room temperature. This method may provide a low cost alter-
native to Armored RNA, while maintaining the advantages of
stability and rapid production.
Another alternative utilizes TMV coat proteins for assembly
of a positive control, with the helical symmetry of the resultant
particle more realistically mimicking the stability of filamentous
viruses. This approach was recently demonstrated for RT-PCR
detection of EBOV.423 To construct the EBOV-TMV mimic,
purified CP from TMV was reassembled around an RNA tran-
script containing an EBOV sequence fragment and a shortened
TMV sequence containing the origin of assembly (OAS) necessary
for TMV assembly (Fig. 18). The EBOV sequence was taken from
a region in the RNA-dependent RNA polymerase gene (L-gene)
that showed homology between all published EBOV sequences.
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Fig. 18 Manufacture of EBOV-TMV. (a) EBOV-TMV is manufactured by disassembly of TMV propagated in N. benthamiana plants into individual coat
proteins that are then reassembled around synthetic RNA transcripts containing EBOV and TMV gene sequences. (b) TEM of negatively-stained wild-type
TMV rods. (c) TEM of shorter EBOV-TMV rods demonstrating successful reconstitution. Scale bar = 100 nm. Reproduced with permission from ref. 423.
Copyright 2016 Nature Publishing Group.
Detection of both the EBOV and TMV sequences was accom-
plished using multiplex RT-PCR with the EBOV-TMV particle.
Aside from the EBOV primer binding sites, the EBOV sequence
was scrambled, therefore posing no threat of infection. Overall,
EBOV-TMV is a scalable construct that could be easily adapted as
a control for EBOV diagnostics, and the technology could be
further applied for mimicking other filamentous viruses.
4.2.4 Nanoreactors. One of the first demonstrations of the
utility of using viruses for the display of biocatalysts was based
on genetic engineering of PVX to display a lipase enzyme.424
Although the catalytic activity of the bound enzyme was lower
than the free enzyme, the study demonstrated the potential
for generating catalytically active nanoparticles that can self-
assemble and be easily propagated. Since then, interest in the
functionalization of virus particles with enzymes has grown.
Coupled with the ability of the viral capsid to self-assemble
around a diverse range of cargo rather than simply nucleic
acids, encapsulation of enzymes to form nanoreactors has
become of increasing relevance to biotechnology.
Initial work with the formation of such nanoreactors looked
at the incorporation of a single enzyme within the capsid.
Through disassembly and reassembly of the capsid, HRP was
encapsulated within CCMV, resulting in an estimated one in
every 130 capsids containing the enzyme and allowing study of
the enzyme at a single-molecule level.425 Comparison of the
activity of encapsulated and non-encapsulated enzyme showed
different signatures due to the time necessary for the dihydrorhod-
amine 6G substrate to diffuse in and the fluorescent rhodamine
6G product to diffuse out of the capsid. Resultantly, product
accumulation and delayed loss of fluorescence was observed
with the spatially constrained HRP. Further investigation with
the confinement of the enzyme Pseudozyma antartica lipase B
(PalB) within CCMV utilized a heterodimeric coiled-coil linker to
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first attach PalB to CP subunits before assembly.426 Through
varying the ratio of CP with and without PalB during assembly,
the average number of encapsulated enzymes could be con-
trolled. Encapsulated PalB had a higher activity compared to
non-encapsulated PalB, which was hypothesized to be due to a
higher enzyme concentration when considering just the capsid
alone. Additionally, there was no effect on the reaction velocity
when varying the number of encapsulated enzymes between one
and four PalB per capsid, which is likely due the presence of
generally only one substrate molecule per capsid, making more
than one enzyme unnecessary for substrate conversion.
Tethered encapsulation was also explored using RNA
aptamers.427 The technique utilized co-expression of Qb CP,
Rev-tagged Peptidase E (PepE) or firefly luciferase, and a
bifunctional mRNA containing an a-Rev aptamer and a Qb
genome packaging hairpin on its two ends for the encapsulation
of the enzyme during expression and assembly. This strategy
resulted in up to 18 enzymes encapsulated within the particle.
The enzymes remained active after encapsulation, and the capsid
was found to offer some protection against thermal degradation,
protease digestion, as well as hydrophobic adsorption. A one-
pot expression-assembly approach has also been utilized for
enzyme tethering within P22, with initial studies investigating
programmed encapsulation of alcohol dehydrogenase D
(AdhD)428 and homotetrameric b-glycosidase enzyme CelB429
using plasmids harboring both genes for the P22 CP and for
its scaffold protein (SP) fused to the enzyme of interest. SPs
associate with the interior of the P22 capsid in such a way that
the enzymes are consequently encapsulated during expression
and assembly of the proteins in E. coli. AdhD encapsulated in
P22 was less active due to enzyme crowding effects but overall
had a similar catalytic efficiency and did not exhibit substrate
inhibition, unlike free AdhD.428 With encapsulation of CelB,
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which is only active in its tetrameric form, incorporation of
multimeric enzymes in P22 was demonstrated.429 Surprisingly,
unlike AdhD encapsulated CelB did not result in loss in activity
or change in substrate affinity. Additionally, embedding the
encapsulated CelB in an acrylamide gel and dehydrating and
rehydrating the gel resulted in over 60% retention in activity,
which could be attributed to substrate diffusion limitations,
therefore presenting the utility of P22 nanoreactors for enzyme
immobilization applications.
A consideration for the use of capsids as a nanoreactor is
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the influence of electrostatics on substrate diffusion into the
carrier.430 MS2 CPs were assembled around negatively charged
alkaline phosphatase (PhoA-neg), and the effect of capsid
mutation adding either negative or positive charges around
its pores was explored. Not unexpectedly, while introduction of
negative charge had an inhibitory effect, additional positive
charge resulted in greater catalysis of the negatively charged
phosphatase substrate. Therefore, engineering of nanoreactors
can be used to control the flux and extent of reaction. Another
benefit of nanoreactors, also seen above with PepE, is the
bestowment of stabilization. As an additional example, encap-
sulation within P22 of phosphotriesterase, which has low heat
tolerance and is prone to hydrolysis, yielded protection from
proteases and desiccation as well as thermal stability with
activity even at 60 1C, making it a more practical option as an
enzyme for combatting various harmful insecticides and nerve
agents.431 The strategy for sequestration of enzymes within P22
during expression is also valuable for the recovery of otherwise
insoluble proteins.432 Some recombinant proteins are trafficked
to inclusion bodies during production in E. coli, making recovery
difficult. a-Galactosidase (GalA) is one such protein that was
studied, and encapsulation within P22 was shown to allow for
successful rescue of properly folded GalA. Encapsulation resulted
in very highly active enzyme, which was hypothesized to be most
likely due to more correctly folded and active enzymes when
produced in this manner.
There is a range of other applications to consider for nano-
reactors, many of which have been demonstrated using P22 viral
scaffolds. For example, it was determined that the immobiliza-
tion of cytochrome P450 (CYP450) inside P22 could be used for its
stabilization and delivery to human cervix carcinoma cells with
retention of substrate conversion activity, which could be further
exploited for enzymatic prodrug therapies.132 In addition, encap-
sulation of NADH oxidase, which predominantly reduces oxygen
to hydrogen peroxide, was demonstrated as a method for bacterial
growth inhibition by triggering oxidative damage in E. coli
cultures.433 Notably, a recent breakthrough in nanoreactor tech-
nology is its use for the catalysis of hydrogen production.434
Sequestration of oxygen-tolerant nickel–iron (NiFe)-hydrogenase
in P22 provided greater stability for the hydrogenase and resul-
tantly, a greater than 100-fold increase in proton reduction activity
over the free enzyme was observed. With the scalable production
of encapsulated hydrogenase through simple fermentation, cheap
and sustainable clean fuel production can be realized.
Further development of viral nanoreactors also explored the
introduction of multiple enzymes tethered to P22.131 By the
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close proximity of enzymes involved in a cascade, the product
of one enzyme can be efficiently taken as the input of the next.
Assessment of enzyme cascade encapsulation was evaluated
with the tetrameric CelB, the monomeric ATP-dependent galacto-
kinase (GALK), and the dimeric ADP-dependent glucokinase
(GLUK), which processes lactose into galactose and glucose,
phosphorylates galactose to form galactose-1-phosphate, and
phosphorylates glucose to glucose-6-phosphate, respectively
(Fig. 19). Using multienzyme GLUK-CelBSP and GALK-GLUK-
CelB-SP fusions, co-encapsulation of two- and three-enzyme
cascades within P22 was achieved. When compared to a 1 : 1
mixture of individually encapsulated CelB-P22 and GLUK-P22
controls, GLUK-CelB-P22 showed greater enzymatic conversion
under inhibitory conditions for CelB. GALK-GLUK-CelB-P22
additionally showed a greater than 2-fold faster turnover rate
than GLUK-CelB-P22, suggesting all three proteins assembled
properly into their active forms and were successfully encap-
sulated. Therefore, enzyme assemblies are of significance in
facilitating the construction of complex metabolic systems.
4.2.5 Agricultural applications. An interesting area that has
just recently been considered is the application of plant viruses
in agriculture. Since their natural hosts for infection are plants,
at first glance it appears counterintuitive to apply such nano-
particles for this particular application. However, the pioneering
study utilized RCNMV for combatting parasitic root nematode
infections and demonstrated that such particles have better
mobility in the soil, therefore improving the bioavailability of
the abamectin pesticide for nematode control.112 Using ligand
gating for infusion (see Section 3.3), the neutrally charged
pesticide could be loaded within RCNMV. In such a manner,
the abamectin cargo is protected against oxidation and can be
released over time. Encapsulated and free abamectin showed
similar efficacies in liquid nematode cultures, but when tested
on infected tomato seedlings, the viral delivery of abamectin
resulted in healthier root growth and reduced root galling
compared to abamectin alone. These results have important
implications for the agricultural industry, where parasitic
nematodes have resulted in astronomical costs on the order of
$118 billion worldwide due to crop damage,435 and future
considerations with noninfectious VLPs could further refine this
strategy of using the naturally evolved mechanisms of such
viruses for cargo delivery to plants.
4.2.6 Plant-based pharmaceutical production. In Section
2.2, we discussed how GlaxoSmithKline, Merck, Medicago Inc.,
and Mapp Biopharmaceutical adopted expression systems
derived from viruses for the production of VLP vaccines and
antibody cocktails. Plants, as used by Medicago Inc. and Mapp
Biopharmaceutical, are not as well known for the expression of
recombinant proteins as the more widely used E. coli and yeast
expression systems, so we will focus on some examples of plant-
based production systems in this section to illustrate some of
their advantages. The utilization of plants as a production
platform has undergone rapid growth, and cost-effective, highly
scalable, and safe production of protein pharmaceuticals with
post-translational modifications can now be achieved using
plant viral vectors.436,437 Due to these advantages in cost and
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Fig. 19 Encapsulation of enzyme cascade in P22. (a) Schematic of P22 nanoreactor assembly where a multienzyme GALK, GLUK, and CelB fusion gene
with an additional SP scaffolding domain is encapsulated in the capsid. The expression of the fusion protein allows the enzymes to form the oligomers
required for activity (tetramer for CelB, dimer for GLUK). The enzymes are colored green, blue, and red, respectively, for the GALK, GLUK, and CelB fusion,
and the CP is shown in gray and SP in purple. (b) Illustration of the metabolic pathways of the enzymes and how they are coupled. Reproduced with
permission from ref. 131. Copyright 2014 American Chemical Society.

production, plant systems offer the potential for rapid pharma-
ceutical development, especially for more impoverished areas.
One of the first examples of transfecting plants with viral
vectors that demonstrated rapid production of a protein relevant
to pharmaceutics utilized an expression vector based on TMV.438
High level heterologous expression of biologically active a-tri-
chosanthin, which can inhibit HIV replication in vitro, was
achieved through insertion of the gene into a TMV plasmid.
By controlling its transcription using a subgenomic promoter,
regulation of the expression of the specific gene could be achieved.
Further research of such N. benthamiana-based expression systems
found that suppression of post-transcriptional gene silencing
(PTGS), the plant’s adaptive immune system, resulted in enhanced
efficiency and was possible through co-expression of the P19
protein of tomato bushy stunt virus (TBSV).439
In parallel, the development of the magnICON ‘‘deconstructed’’
TMV vector system440 has resulted in advancements in the produc-
tion capacity without need for PTGS suppression.441 The magnICON
system involves engineering DNA modules for replication, the gene
of interest, and recombination, with the advantage of being easily
modifiable using Gateway technology, a universal system for
cloning. The modules are then delivered to plants by Agrobacterium
and result in transient gene expression and production of viral RNA
replicons that can replicate autonomously.440 Furthermore,
through ‘‘magnifection,’’ or weak vacuum infiltration of the
plants immersed in the Agrobacterium suspension, transfection
at a large scale can be quickly achieved without the need of the
CP gene or the wait for systemic plant movement.442 A large
variety of biologically relevant pharmaceutics have been produced
using magnICON, including VLP vaccines,443,444 antibodies,445,446
plague antigens,447 cytokines,448 and growth hormones.449
The TMV RNA-based overexpression (TRBO) vector is another
replicon system that has been developed and has shown promise
for high-level protein expression, with greater yields than demon-
strated with the aforementioned P19-enhanced transient expression
(Fig. 20).450 Essentially, the TRBO vector is a 35S promoter-driven
TMV expression vector with the CP gene removed. The CP deletion
resulted in a higher efficiency of recombinant protein expression,
which was demonstrated for a range of proteins, including GFP,
adenosine kinase, the 10th domain from human fibronectin, and
some proteinases. Another study also demonstrated the usefulness
of the TRBO vector for production of R8, a chimeric allergen derived
from dust mites that could be applied for asthma diagnosis or
immunotherapy.451
Similar expression systems have been derived from plasmids
based on PVX and other potexviruses. Complete PVX constructs

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Fig. 20 Quantitative analysis of GFP expression with TRBO vector.
Fluorescence (in mg GFP per g infiltrated tissue) was measured from
N. benthamiana leaves after agrobacterium infiltration (top). Leaves were
also imaged under UV light (bottom). The labels in the figure indicate the
plasmids used for transformation, and the results indicate the superior
expression of protein with TRBO vector compared to previous expression
vectors, even with P19 enhancement. pJL-24 is a previous iteration of a
35S promoter-driven TMV-based expression vector that included the
expression of all the TMV genes in addition to the gene insert, pJL3:P19
is a plasmid for the expression of the RNA-silencing suppressor protein
P19, pJL TRBO-G is a GFP-expressing TRBO vector, and p35S:GFP is a
plasmid with GFP expression under the control of a 35S promoter.
Reproduced with permission from ref. 450. Copyright 2007 American
Society of Plant Biologists.
can be used for the expression of recombinant proteins along-
side PVX CPs through insertion of an internal ribosome entry
site, which allows for initiation of translation in the middle of
an mRNA, between the two genes.452 Similar constructs that
instead have an intervening FMDV 2A cleavage sequence can be
used to create proteins expressed as a fusion to the CP.453 While
larger inserts tend to result in genetic instability and loss of the
gene of interest,454 the combination of introducing a hetero-
logous subgenomic promoter from bamboo mosaic virus and
deleting a portion at the N-terminus of the CP was found to be a
successful strategy to increase transgene expression stability.455
Deconstructed vectors have also been used for PVX-based
expression systems. For example, through replacement of the
CP gene and the triple gene block (TGB) that encodes movement
proteins, efficient production of the gene insert can be achieved.456
Suppression of PTGS helped to improve yields, with transient
co-expression of P19 from TBSV or HC-Pro from tobacco etch virus
(TEV) resulting in a 44% and 83% increase in gene expression,
respectively.457 Amplicon-plus Targeting Technology utilizes this
benefit of gene silencing suppression to enhance production.458
This technology involves using a transgenic TEV-B tobacco line that
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expresses the suppressor gene that is then infected with a PVX
amplicon containing the gene of interest. Successful production of
a highly labile L1 vaccine protein from canine oral papillomavirus
was achieved when this system was used for the expression of
the recombinant protein fused to a chloroplast targeting peptide.
Coupling of PVX and TMV viral vector systems has also been
evaluated and was demonstrated to be a useful approach for
resolving complications from competition between multiple
replicons in the same cells.446 With the noncompetitive viral
systems, expression of assembled oligomeric proteins can be
accomplished, such as for construction of full-length IgG with
its heavy and light chains.445,446
Some other examples of potexvirus vectors include vectors
based on plantago asiatica mosaic virus (PlAMV)459 and foxtail
mosaic virus (FoMV).460 The recombinant PlAMV vector was
found to have greater genetic stability and longer retention of
the inserted gene compared to PVX, likely due to stronger RNA
silencing suppression activity found from the first movement
protein in its TGB.459 On the other hand, the FoMV-based FECT
vector series utilized deconstructed plasmids in which the CP
and TGB were deleted and required co-expression of the P19
suppressor of PTGS.460
Moving on to other systems, CPMV-based expression systems
have also shown great versatility for the enhanced expression of
a large range of proteins, including antibodies,461 human gastric
lipase,462 and VLP vaccines (such as BTV VLPs discussed in
Section 4.1.3).230 CPMV is a bipartite virus with RNA-1 providing
replication and protein processing capabilities and RNA-2 coding
for movement and coat proteins. By altering the shorter RNA-2
through removal of the movement and coat proteins and appending
the gene of interest, expression of foreign genes can be achieved
through inoculation of plants with constructs of both RNA-1 and
the modified RNA-2.463,464 Agroinfiltration of just the RNA-2
construct with the TBSV P19 suppressor of silencing was found
to overcome the necessity for RNA-1, and additional elimination
of the second start codon in the 5 0 untranslated region (UTR) of
RNA-2, located at position 161, resulted in hypertranslation of the
downstream protein, likely due to AUG 161 being inhibitory for
overall translation.465 With this discovery, high protein expression
could be achieved without restrictions on insert size and without
the need for RNA-dependent RNA polymerases.
The hypertranslatable CPMV or CPMV-HT system described
above was packaged into pEAQ expression vectors for easy
recombination, where the gene of interest can be inserted
between the modified 5 0 UTR and the 3 0 UTR of RNA-2, and
with P19 expressed from the same plasmid.466 The pEAQ
vectors have broad applications, but perhaps one of its greatest
advantages is the production of VLPs for vaccines. Medicago
Inc. uses CPMV-HT for its large-scale production of enveloped
influenza H5 VLPs, with observed budding from the plasma
membrane resulting in envelopment and similar structural
characteristics to influenza viral particles.467 eCPMV can also
be produced using the pEAQ system, where the necessary
proteins for CPMV formation, namely its VP60 CP precursor
and a 24K proteinase, could either be expressed from the same
vector or from two CPMV-HT vectors to form the mature eCPMV
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particle devoid of any nucleic acid encapsulated.468 Since the
pEAQ vector is nonreplicative, levels of expression of multiple
proteins can be controlled through co-infiltration of appro-
priate concentrations of the expression vectors. In such a
manner more complex VLPs such as BTV VLPs consisting of
up to four different proteins can be assembled.230
Our last examples of plant viral expression vectors come
from geminiviruses, which are small ssDNA viruses with
twinned capsid morphology. The in-plant activation (INPACT)
expression platform is notable for its use of a split-gene cassette
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in stably transformed plants.469,470 The INPACT cassette is
based on a deconstructed tobacco yellow dwarf virus (TYDV)
genome and consists of two components: (1) the gene of
interest split into two exons with flanking large intergenic
regions (LIRs) and a small intergenic region (SIR) in between,
and (2) Rep and RepA activator genes required for replication
and activation of recombinant protein production that are
inducible by ethanol. Binding of Rep to a site within the LIR
initiates rolling circle replication of the replicon system. With
ethanol induction, protein production is separate from plant
growth, allowing high levels of protein expression that could be
cytotoxic or inhibit plant development.
Another method uses a single-vector DNA replicon system
based on bean yellow dwarf virus (BeYDV) and is exemplified by
the production of oligomeric monoclonal antibodies protective
for EBOV.471 The plasmid contains two tandemly linked replicons
for the heavy and light chains and only requires the SIR, LIR,
and Rep/RepA viral components. The system resulted in non-
competing replicon amplification and protein expression, with
efficient assembly of the IgG tetramer. As a final example, pRIC
is a similar BeYDV-derived autonomously replicating vector472
that presented an advancement to a previous high expressing
but non-replicating pTRAc cassette.473 The pRIC replicon gene
vector was created by the addition of the SIR, LIR, and Rep/RepA
genes and resulted in gene amplification up to 2 orders of
magnitude and up to 7-fold greater production of HPV-16 major
CP L1 and HIV-1C p24 subunit vaccine antigens compared to
pTRAc.472
There are clearly many options for quick and high yielding
recombinant protein production in plants. Although MagnICON
has been used widely in the past, its utilization of multiple
modules likely detracts from its efficiency, and the development
of other systems using single constructs may lead to greater
popularity. The main applications for plant-based production
have been for antibody and vaccine production, but pharma-
ceutical proteins of all types can also be produced with the above
techniques.
4.3 Energy and nanostructured materials
The design and development of devices with nanoscale features
open the door for novel and more efficient ways to capture,
store, and transfer energy. Since viruses are self-assembled
from coat proteins into nanoscale structures, and the protein-
based building blocks also show an inherent propensity to self-
assemble into higher-order hierarchical assemblies,124,474–479
they provide an ideal building scaffold for the design of
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nanostructured materials. The versatility of hybrid virus-based
materials in energy sciences and applications has already been
recognized. Examples are highlighted in the following sections
of the functionalization of virus-based materials to yield energy-
relevant materials such as light harvesting systems, plasmonic
metamaterials, and energy and data storage systems.
4.3.1 Principles of self-assembly: wires, sheets, and arrays.
A hurdle to the production of mesoscale nanostructured materials
is the availability of high-precision manufacturing technologies
that facilitate large-scale assembly while also providing spatial
control at the 1–100 nanometer level.480 Top-down approaches,
derived from technology implemented by the computer industry,
have progressed to provide tighter control of feature dimensions
with impeccable reproducibility. To program feature components,
they rely on lithographic fabrication, such as photolithography,481
microcontact printing (or soft lithography),482,483 block copolymer
nanolithography,484,485 nanoimprint lithography,486 and scanning-
probe or dip-pen lithography.487,488 Although top-down
approaches facilitate extraordinary reproducibility in the writing
of nanoscale features at the centimeter size scale, the technology
is highly specialized and feature sizes are still limited.
On the other hand, bottom-up approaches seek to achieve
directed and controlled assembly of individual components
into hierarchical architectures, and they more closely mimic
biological systems, cells, and organisms, which can orchestrate
complex energy conversion functionalities. Developments in
the art and science of self-assembly have made tremendous
contributions to the 3D organization of composite materials,
rendering high precision manufacturing of energy-relevant
biomolecular and inorganic materials possible. For example,
DNA-based ‘‘programming’’ exploits the sequence-specificity
of base pairing to precisely position materials in 2D and 3D
space.489–499 Chemical programming of hierarchical structures
has also been devised, as seen in the synthesis of branched
dendrimer systems.500–505 Other methods include the use of
synthetic block copolymers506 or the application of orthogonal
pairs of coiled-coil peptides507,508 to induce self-assembly of
nanoparticles. It is clear that self-assembly holds great
potential for the nanomanufacturing of mesoscale materials
through simple solution-based bottom-up synthesis, and virus-
based self-assembly is one such approach that presents several
unique advantages.
For example, high aspect ratio virus particles, such as the
plant virus TMV and bacteriophage M13, form excellent
biology-derived scaffolds for the templating and synthesis of
inorganic matter to produce nanowires at the mesoscale.282,509–511
In the case of TMV, mineralization can be achieved both in its
interior channel119 and around its exterior surface,282 leading to
explicit control of the width of the wires. By coupling viral
particles with mineralization techniques, semiconducting, super-
conducting, and insulating nanowires can be formed, resulting in
hybrid materials with properties of interest to energy sciences and
the electronic industry.
High-order assemblies bridging the nano-to-mesoscale can
also be achieved with self-assembly. For instance, TMV building
blocks can be specifically directed to assemble end-to-end
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(or head-to-tail) or side-to-side when exposed to appropriate
bathing conditions.512,513 At acidic pH, TMV rods tend to align
head-to-tail and form long wires due to hydrophobic inter-
actions between the dipolar ends of the TMV rod.514 The 1D
TMV wires can be further stabilized and condensed to form
bundles with the assistance of aniline through in situ polymeri-
zation of polyaniline.478 By additionally incorporating DNA
hybridization, progress has also been made toward greater
control in the specific ordering of viral particles when assembled
end-to-end.515 To accomplish this, the ends of M13 phage were
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functionalized with different DNA oligonucleotides in such a
manner that introduction of the appropriate hybridizing oligo-
nucleotides led to the assembly of ordered phage trimers (Fig. 21).
Although this work investigated controlling the sequence of
phages labeled with different fluorescent dyes, in the future it
can be further extended for the formation of heterofunctional
multiphage structures with distinct moieties that impart more
complex functionalities.
The high aspect ratio structures formed by TMV and phages
M13 and fd have also long been used to produce and study
liquid crystalline arrangements, which may find applications in
next-generation electronic displays. To yield liquid crystalline
assemblies, in-solution mixing protocols have been developed
to drive the alignment and spatial organization of the protein-
aceous building blocks. A classical approach makes use of
concentration gradients such that a nematic liquid crystalline
phase is generated at a critical concentration.516 The onset of
ordering can be explained by the Onsager theory for isotropic–
nematic phase transition, which states that there is competition
between translational and orientational entropy, leading to
higher densities favoring the nematic phase in order to confer
greater translational entropy.517,518
Filamentous viruses are able to form the same mesophases
exhibited by other rod-shaped liquid crystal materials: nematic,
cholesteric, smectic A, and smectic C, and they can exist
interchangeably.477,519 For example, M13 phage-based liquid
crystals present a nematic phase at low concentrations. As the
concentration increases, a cholesteric liquid crystalline phase is
observed. Finally, at high concentrations, the system exhibits
a smectic liquid crystalline phase. Furthermore, based on the
anisotropic nature of the filamentous viruses, external stimuli
Fig. 21 Ordered assembly of phage trimer by DNA hybridization. (a) Diagram of design of multiphage structure using DNA hybridization. pIX protein
displaying DNA sequence A and pIII protein displaying sequence B are linked by complementary sequence C. Similarly, pIX and pIII proteins displaying
sequences D and E, respectively, are linked by complementary sequence F. (b) Fluorescence microscopy image of phages after assembly illustrated the
specific arrangement of the phages. Reproduced with permission from ref. 515. Copyright 2013 American Chemical Society.
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such as electric and magnetic fields can be applied to tune
the ordering, therefore allowing alignment of the whole
sample.13,520,521
Ordering can also be achieved at liquid/liquid and liquid/
solid interfaces. For instance, when the oil/water interface
between perfluorodecalin and water was explored, it was found
that TMV rods aligned parallel to the interface at low concen-
trations but aligned normal to the interface at high concentrations,
which could be attributed to a compromise between maximizing
interfacial interactions and minimizing electrostatic repulsion
between the rods.475 Parallel alignment of TMV can also be
achieved through assembly at interfaces between buffer and a lipid
monolayer supported on a submerged hydrophobic substrate, with
the inclusion of Ca2+ ions helping to screen Coulomb repulsion
between the particles.522,523 Another possibility for the formation
of assemblies of high aspect ratio viruses is through mixtures with
spheres; the increased free energy at the interface favors the
assembly of uniform structures.524,525 This was observed in a study
where isotropic, nematic, lamellar, and crystalline phases were
obtained through the modulation of the concentration of TMV,
the concentration of BSA or PEG spheres, and the ionic strength.
Addition of divalent metal cations has also been shown to
induce the formation of ordered aggregates of TMV. Precipi-
tates of TMV with nematic liquid crystal properties were formed
with the addition of ions such as Cd2+, Zn2+, Pb2+, Cu2+, and
Ni2+, an effect that did not extend to the alkaline earth metals
Ca2+ and Mg2+.526 Furthermore, highly ordered, optically bire-
fringent films were formed when the aggregates were dried on a
glass surface. It is hypothesized that TMV contains low-affinity
metal binding sites that induce its crosslinking in the presence
of the metal ions. While beneficial for liquid crystal applications,
this behavior poses an important consideration when formation of
well-dispersed biomineralized TMV is desired in the formation of
hybrid materials. For example, achieving conditions for dense
copper deposition without aggregation of the TMV templates is a
challenge.527 Nevertheless, exciting new properties may be possible
as a result of particle assembly. Assembly of TMV in the presence
of Ba2+ is of particular interest, as instead of the liquid crystalline
ordering found for other divalent metal ions studied, the TMV
crystallized into a hexagonal superlattice that may have interesting
electronic properties.528
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In addition to in-solution techniques, electrospinning,
wet spinning, and dip coating have been applied to generate
long-range ordered filamentous virus-based films.333,529 For
example, varied chiral liquid crystalline M13 phage films were
obtained with the use of dip coating methods.333 By carefully
adjusting phage concentration, pulling speed, bathing conditions,
M13 surface chemistry, and surface chemistry of the solid
support, supramolecular M13 films with nematic orthogonal
twists, cholesteric helical ribbons, or smectic helicoidal nano-
filaments could be produced. These M13 arrays exhibited
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onto a functionalized surface primed to interact with the virus-
based nanoparticles.537 Toward free-standing films, we have
recently demonstrated the development of detachable meso-
porous films, using a combination of nanosphere lithography
and electrodeposition to form nanopatterned, conducting virus–
polymer arrays (Fig. 23). To accomplish this, a hexagonally close-
packed array of polystyrene (PS) latex microspheres was created
using colloidal or nanosphere lithography, yielding a mesoporous
architecture. A conducting poly(pyrrole-co-pyrrole-3-carboxylic
acid) film was then electrochemically polymerized in the inter-

unique optical and photonic properties such that the films
showed iridescence when illuminated, which has potential
applications as reflectors or displays. Another potential area
of application lies in colorimetric sensing. For example,
matrices formed using dip coating of M13 displaying binding
peptides for the explosive TNT showed significant enough
structure changes in the presence of TNT vapors that levels
down 300 ppb could be selectively distinguished (Fig. 22).395
Another area of interest is the use of viral building blocks for
the patterning of surfaces, which can be accomplished using a
variety of strategies, including conjugation chemistries530,531
and electrostatic interactions.479,532–536 The patterning of virus-
based nanobuilding blocks has also been demonstrated using
photolithographic and microcontact printing strategies, where
a hard elastomeric pattern is ‘‘inked’’ with virions and stamped
stitial voids between the PS beads by cyclic voltammetry.
Following PS template removal, CPMV particles were deposited
atop the conducting polymer film through electrostatic adsorption
and hydrogen bonding. The resultant Janus polymer–virus film
was found to be robust and stable, allowing it to be electro-
chemically delaminated from the substrate.
Other methods for VNP immobilization onto a surface have
utilized DNA hybridization, a powerful technique proven for
effective guided assembly of materials in 3D space.489–499 DNA
hybridization facilitates controlled and reversible assembly
through adjustment of sequence specificity, temperature, or
chemical cues (e.g. access of free nucleotides). The TMV building
block is one example where nucleic acid hybridization was applied
for the programming of materials.538,539 Here, TMV underwent a
mild disassembly protocol to expose the 50 end of its genome,

Fig. 22 Phage litmus for TNT detection. (a) Phages genetically engineered with binding peptide for TNT were self-assembled through dip coating with
varying pulling speeds to form bundled structures that resulted in colored matrices. (b) Structural changes upon TNT binding resulted in color changes
that can be detected using an iPhone-based analysis system down to a level of 300 ppb, with dashed red line showing the sensitivity limit. (c) Images and
processed fingerprints from the colorimetric sensor after exposure to MNT, DNT, and TNT demonstrated selective sensing of TNT over the similar
molecules. (d) Principal component analysis of the color changes further verified selectivity. Reproduced with permission from ref. 395. Copyright 2014
Nature Publishing Group.

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Fig. 23 Creation of free-standing Janus mesoporous virus film. Topo-
graphical tapping mode AFM images illustrate the initial formation of
close-packed PS microspheres (a), partial removal of PS spheres (b) after
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patterned poly(pyrrole-co-pyrrole-3-carboxylic acid) electropolymerization
(c), and a non-patterned film (d). (e) Optical microscope image (480 mm by
360 mm) after overlaying CPMV on the patterned polymer through electro-
statics and delaminating the film to create a free-standing film. Insets show
topographical AFM images at indicated points in the film.
which could then be used as a code to guide the assembly of
vertical TMV arrays, either directly with the corresponding
complementary oligonucleotides displayed on the surface,538
or through an intervening DNA linker with sequences specific
for both the TMV and the surface.539 As an alternative nucleic
acid-based method, RNA templates containing the TMV OAS
can be patterned onto surfaces, giving a cue for the in situ
formation of TMV arrays from the surface up when purified
TMV coat proteins are added.540,541
Icosahedrons also make excellent building blocks for self-
programmed assembly of crystalline arrays and lattices. Two-
dimensional virus arrays of CPMV have been formed through
a drop-and-dry method on a mica substrate, with packing
symmetry controlled by modulating the PEG surfactant concen-
tration.542 The organization of the rhombic and hexagonal
closed packed structures formed can be attributed to both
steric requirements as well as the charge distribution around
the capsid surface. Another approach utilizes interfacial
adsorption of viral particles under a cationic lipid monolayer
that is formed at the surface of an aqueous solution.543,544
Based on studies with CPMV and TYMV, varying 2D array
architectures such as rectangular, hexagonal, and rhombic
lattices were achieved using this method.
For 3D crystals, a standard technique applied is depletion
condensation, in which PEG is a common condensing agent
used for the induction of virus crystallization in bulk.545
Toward the programming of binary materials and photonic
crystals, the organization of compact structures of Qb phage
and gold nanoparticles through DNA interconnectors was
reported, and the binary lattice was shown to form a NaTl
lattice structure that contained interpenetrating organic and
inorganic diamond lattices.546 Assembly of viruses can also
be mediated by electrostatic self-assembly,547 and organization
of particles into crystalline lattices using this method has been
demonstrated through the introduction of dendrons and
dendrimers.548–550 More recently, binary AB nanoparticle super-
lattices were achieved using icosahedral CCMV and either gold
nanoparticles551 or avidin.552 The patchy, but symmetrical, charge
distribution on the virus capsid surface enables the formation of
binary cubic structures. The authors have also recently extended
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these concepts to the non-viral protein cage ferritin, yielding
photoactive biohybrid crystals with phthalocyanine dyes.553 More
specifically, face-centered cubic crystals up to 100 microns in
size were obtained in which the phthalocyanines maintained
their native properties, suggesting that such materials could
find applications in photodynamic therapy, water treatment,
diagnostic arrays, and as an oxidant in organic synthesis.
4.3.2 Data storage and battery electrodes. The programm-
ability of the viral scaffold, combined with its propensity to
self-assemble into 3D hierarchical architectures, has led to its
application toward devices such as battery electrodes, digital
memory devices, and energy storage devices. As discussed and
illustrated in earlier sections, viruses offer a framework for
ligand and peptide display. As a result, they can facilitate the
precise deposition of inorganic and organic materials such as
metals,67,533,554–556 metal oxides,511 semiconductors,557 graphene,558
and carbon nanotubes,559 all of which are energy-relevant materials.
To achieve digital memory, virus hybrids can be created
such that they exhibit conductance switching behavior.560,561
One method to fabricate such a device involved decorating TMV
with Pt nanoparticles and using them to form a composite layer
in a PVA matrix sandwiched between two electrodes.560 As a
result of charge trapping in the nanoparticles, the device
exhibited bistable low conductance OFF and high conductance
ON states, which were observed with the application of a
reverse bias below 2.4 V or a forward bias above 3 V,
respectively (Fig. 24). In a similar manner, CPMV decorated
with semiconducting quantum dots also demonstrated bistable
ON/OFF electrical behavior.561 Additionally, for both systems, it
was shown that process was reversible and could undergo
repeated cycles of reading and writing. Although the maximum
limit was found to be around 400 cycles, suggesting the need
for further refinement, these investigations have established
the use of biomaterial hybrid systems for memory storage and
motivate the exploration of future possibilities.
Some early work with scaffolds formed by CPMV investi-
gated the positioning of redox-active species, such as ferrocene
and viologen, on the viral template.562,563 The multivalent redox-
active nanoparticles exhibited simultaneous multielectron transfer,
indicating that the multiple redox centers are independent and
behave as essentially non-interacting redox units. Such materials
may be envisioned as multielectron reservoirs and as nanoscale
electron transfer mediators in redox catalysis or amperometric
biosensors.
Another electrical application for viruses involves their
utilization as battery electrodes. Research has been performed
for the creation of both cathodes and anodes based on M13 as
well as TMV templates.510,555,556,564–567 Using self-assembly
of the viruses, fabrication of high performing small battery
electrodes was made possible. Early studies utilized M13 to
grow cobalt oxide nanowires that served as the active anode
material, and they demonstrated that virus network formation
is a versatile method that could be applied for the formation
of electrodes on both rigid and flexible substrates with
full electrochemical functionality and greater capacity.510,564
Additional hybridization of the nanowires with Ni nanoparticles
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Fig. 24 TMV-based digital memory device. (a) TEM image of TMV with approximately 10 nm-sized Pt nanoparticles uniformly attached. (b) I–V curves of
device created with an active layer derived from the TMV–Pt nanowires (illustrated in inset). The curves demonstrate the conductance switching behavior
of the device, with a switch to the ON state during the first bias scan (blue filled circles) at 3.1 V, stability in the ON state with the second scan (blue open
circles), and a return to the OFF state during a reverse scan (blue squares) at 2.4 V. On the other hand, devices made from only TMV (red triangles) and
only Pt nanoparticles (red diamonds) showed no conductance switching behavior. Reproduced with permission from ref. 560. Copyright 2006 Nature
Publishing Group.

was found to enhance efficiency and cycle life.567 When M13 was
instead used for biotemplating of manganese oxide to serve as a
cathode for lithium–oxygen batteries, improved capacity of
porous networks formed by the virus was also observed.565 On
the other hand, TMV electrode development utilized its ability
to assemble vertically on a substrate to form ‘‘nanoforests’’
with high surface area and room to accommodate for volume
expansion.555,556 Engineering of the TMV anode involved Sn
nanoparticle deposition on Ni-coated TMV, which was then
further coated with carbon. In such a way, Sn aggregation that
usually occurs when it expands during performance in sodium
ion batteries was suppressed.555 Alternatively, the TMV-based
cathode was designed for integrated lithium ion batteries, and it
was formed by coating TMV-templated Ni nanorods with Ti,
LiFePO4, then carbon.556 Aside from excellent electrochemical
performance, both TMV electrodes exhibited high mechanical
and electronic integrity.
An additional M13-templated electrode example that is of
note is one that utilized iridium oxide as the anodic material.511
IrO2 is exciting because it is an electrochromic material, which
experiences color change through electrical potential application,
a useful property for paper-like display devices. By chemically
attaching gold nanoparticles to M13 before mineralization of
IrO2, IrO2–Au hybrid nanowires were formed to enhance electron
mobility. Porous electrodes made of these nanowires exhibited
remarkable switching times of 35 ms for oxidation/coloration and
25 ms for reduction/bleaching, which is promising for applications
requiring fast electrochromics.
Further energy materials that can be engineered with viral
scaffolds for energy storage and generation devices include
graphene sheet conducting frameworks and Au–Pt core–shell
catalytic nanowires.558,568 In the first case, M13 was used to
stabilize graphene sheets in aqueous media stabilized to prevent
the spontaneous aggregation between individual graphene sheets.558
M13 also served as a viral template to facilitated the nucleation
of bismuth oxyfluoride, a conversion reaction cathode material,
to form graphene/bismuth oxyfluoride nanocomposites that
could be used as a conducting framework for energy storage
with improved electron transfer kinetics. In the case of the
catalytic nanowires mentioned above, M13 was used as a tem-
plate for mineralization of Au followed by Pt, a design with fuel
cell applications.568 With the Au core as a co-catalyst to oxidize
and remove carbon monoxide, the Pt nanowires could be used to
oxidize ethanol with less vulnerability to any such carbonaceous
species that are formed. This strategy demonstrated excellent
catalytic activity, and it additionally has advantages of simple
scale-up synthesis, greater durability of the Pt catalyst, and lower
cost due to the presence of M13 reducing dead volume of Au in
the core.
As a final example of the energy generation potential of
phages, liquid crystals formed from M13 phage have been
shown to exhibit piezoelectric properties.569 Glutamic acid
residues were introduced at the N-terminus of the pVIII coat
proteins in order to create a greater net dipole moment
directed from the N-terminus to the positively charged
C-terminus, which resulted in an enhancement in the effective
piezoelectric coefficient of the phage films. Self-assembled
phage monolayers with nematic structure and phage multi-
layers with smectic structure all demonstrated piezoelectric
behavior, with the response increasing in accordance to
increasing film thickness until saturation occurred at around
a thickness of 100 nm. The investigation further demonstrated
easily fabrication of multilayer phage films through sponta-
neous ordering after dropcasting, and the energy output from
mechanical pressure of the film was sufficient to turn on a
liquid crystal display. Overall, this phage-based technology
presents an opportunity for environmentally friendly energy
generation.
4.3.3 Light harvesting and catalysis. Solar energy is another
source of green energy. Light harvesting systems, such as found in

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the photosynthesis machinery of plants, enable the conversion of

light into energy.570 Chromophores transport energy using
Förster resonance energy transfer (FRET), whereby electrons
are excited upon photon absorption by one chromophore then
transfer their energy to another chromophore as they undergo
relaxation through further photon emission. The excitation
energy gets transferred in a cascade from donors to acceptors
when the energy levels of absorption and emission match. The
photosynthesis system of plants contains precisely spaced
arrays of chromophores that facilitate light harvesting and
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conversion into energy and is one of nature’s most sophisticated

energy conversion complexes.570
Synthetic light harvesting systems are of great interest in
basic energy science and technology development for imple-
mentation in solar panels and other photovoltaic devices. Like
photosynthetic machinery, viral capsids provide a means to
precisely position chromophores with spatial control at the sub-
nanometer level. Recognizing this engineering design space,
a few examples have been published using the self-assembling
TMV scaffold.68,571,572 In brief, TMV coat proteins were covalently
labeled with either a donor or an acceptor from a FRET pair and
then assembled into disk or rod structures at a controlled ratio of
donors to acceptors. Through assembly, the chromophores could
be positioned in close proximity at tunable distances in such a
way to achieve the most efficient FRET. Similarly, M13 displaying
zinc porphyrins has also shown promise as a light harvesting
antenna, and FRET was observed in which the porphyrin served
as both a donor and an acceptor for exciton migration along the
virus.573
A very recent study has revealed groundbreaking work that
further enhances the efficiency of energy transport in light
harvesting devices.574 For the first time, experimental evidence
was found that demonstrated a super-Förster regime where
quantum coherence and classical incoherent mechanisms interact
to enhance exciton transfer efficiency compared to what can be
found with classical FRET. Genetic engineering of M13 was utilized
to regulate the distances between chromophore binding sites to
be either around 33 Å for the ‘‘Classical Förster’’ clone or 10 Å for
the ‘‘Super-Förster’’ clone, a distance short enough for increased
coupling strength but not too short to experience complete
quenching. Fluorescence measurements from the virions modified
with a range of donor-to-acceptor ratios of chromophores demon-
strated the superior behavior of the Super-Förster clone (Fig. 25).
Additionally, there was a 68% enhancement in exciton diffusion
length, an exciting outcome demonstrating enhanced exciton
transport that has previously only been theorized.575
As another part of the light harvesting process, filamentous
viruses have also been employed for electron harvesting as
nanowire-based photoanodes due to their ability to form porous
networks with high surface area. Previous work has investigated
the implementation of M13 in dye-sensitized solar cells
(DSSCs).559,576–578 The design of DSSCs involves the presence
of a dye or photosensitizer for light absorption that, when
excited, transfers the energy into the conduction band of a
TiO2 photoanode, where it then diffuses to a current collector
such as fluorine-doped tin oxide. By using M13 as a sacrificial
Fig. 25 M13 Super-Förster clone for enhanced exciton transport. (a) M13
Classical Förster (left) and Super-Förster clones (right) showing engineered
binding sites for chromophores (N-terminus in blue, pre-existing lysine
residue in green, and inserted lysine residue in orange) and the networks
for energy transport between the residues. Blue arrows show classical
incoherent exciton hopping, while red ellipses indicate exciton delocalization.
(b) Experimental data of fluorescence per acceptor to donor-to-acceptor
ratio of the Super-Förster clone is best matched by numerical simulations
based on Super-Förster theory and decohered quantum walk (QW) rather
than based on classical Förster. Reproduced with permission from ref. 574.
Copyright 2016 Nature Publishing Group.
template for photoanode formation, it was shown that a highly
interconnected porous TiO2 structure could be achieved for
greater charge transport efficiency. Strategies to create a porous
TiO2 network included using electrostatic interactions for
complexation of TiO2 nanoparticles and M13 viruses,576 layer-
by-layer phage deposition before TiO2 nucleation,577 and cross-
linking of the phages by glutaraldehyde to form a hydrogen
before TiO2 nucleation.578 After removal of the sacrificial viral
scaffold, the photoanode was found to possess improved
electron diffusion properties. With additional incorporation
of gold nanoparticles, the dye molecules were found to also
experience greater light absorption through localized surface
plasmon resonance, therefore further improving light harvesting.578
As another step toward improvement of DSSCs and other photo-
voltaics, stabilization of SWNTs by M13 through non-covalent
binding before biomineralization of TiO2 was used for successful
incorporation of the SWNTs in the photoanode.559 Due to the high
electron mobility of the SWNTs, a high power conversion of 10.6%
was observed with this strategy.

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Chem Soc Rev
Another example of M13-based photovoltaic cells involves
the mineralization of perovskite nanocrystals, which have
unique catalytic, electric, and magnetic properties.557 In parti-
cular, M13 was used as a template for strontium titanate (STO)
and bismuth ferrite (BFO) mineralization. The study presented
the first report of the photovoltaic properties of BFO nano-
particles, which showed effective absorption of visible light but
a solar power conversion efficiency of only 0.17%. On the other
hand, the STO nanowires exhibited photocatalytic water
reduction properties, and they could be used for the efficient
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hydrogen production under both UV and visible light. Addi-
tional research in this area investigated the improving the
performance of STO nanowires under visible light.579 Whereas
dye-sensitization was required in the previous study,557 a new
fabrication method was developed that utilized ammonia gas
treatment to tune the optical absorption of the STO nanowires,
which was successful for the manufacture of visible-light active
photocatalysts.
Light conversion has also been explored with TMV.554 In the
study, TMV was arranged vertically on a gold-coated indium tin
oxide (ITO) surface, and the particles were then coated with nickel,
ITO, followed by CuO; the ITO served as a current collector, while
the CuO served as a photocathode. At a high enough TMV density,
the film was found to form an antireflective surface due to its high
surface area and surface roughness, which is beneficial for greater
photoelectrochemical cell efficiency. Various CuO thicknesses were
investigated, and a thickness of 520 nm was found to produce one
of the highest photocurrent densities reported for CuO systems its
size. Overall, the properties of this virus-templated surface bring
much promise for future photoelectrochemical applications, such
as catalytic water conversion.
4.3.4 Plasmonic metamaterials. Plasmonic nanostructures
have already led to breakthroughs in optics and nano-
photonics,580–583 as well as in biotechnology and bio-
medicine.584,585 For nanometer-sized noble metal particles,
the de Broglie wavelength of the valence electrons is of the
same order of magnitude as the size of the particle, and as a
result quantum size effects may be observed. The characteristic
plasmon resonance bands that lead to extraordinary metal
enhancement effects arise due to the oscillation of the valence
electrons at a collective oscillation frequency.585,586 Knowing
this, nanostructures can be designed and organized such that
their plasmonic bands are finely tuned. For applications in the
body, a plasmonic range in the NIR imaging window where
light can penetrate several centimeters into the tissue is desirable.
The substantial absorption and scattering properties of plasmonic
nanostructures can then be used as contrast agents for optical
imaging, such as optical coherence tomography587,588 and photo-
acoustic imaging.589 As a demonstration of a potential application
of plasmonics, sensing using viral plasmonics has been achieved
by using M13 phage loaded with gold nanocubes for SERS.590
Alternatively, the plasmonic nanostructures also have applications
in a therapeutic context due to the transformation of the absorbed
light into heat,591,592 which could be applied for the thermal
ablation of tumors or the controlled release of drugs.593,594 Use as
‘‘theranostic’’ reagents, where diagnostic imaging and therapeutic
4112 | Chem. Soc. Rev., 2016, 45, 4074--4126
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Review Article
functions are merged, presents another option.595,596 Beyond
healthcare, it should be noted that plasmonics can also be
applied to many disciplines, including nonlinear plasmonics,
electronic transport, local heating, biosensors, quantum optics,
and metamaterials.597
Virus-based plasmonic metamaterial synthesis was first
demonstrated with BMV shells assembled around gold nano-
particle cores, where 3D crystals formed by the particles exhi-
bited an optical transmission spectrum indicative of multipolar
plasmonic coupling between adjacent gold cores.124 More recently,
MS2 phages were functionalized with internal gold nanoparticles
and external fluorophores, with the intervening distance controlled
using oligonucleotide hairpins (Fig. 26).174 Enhancements in
fluorescence intensity and corresponding decreases in lifetime
were observed, and the results correlated well with expectations
of the effect of the separation between the plasmon and gain
materials.598–600 In the reverse configuration (internal fluorophores
and gold nanoparticle antennas), fluorescence enhancement was
also observed, and it was found to be a function of nanoparticle
size and the separation between the plasmon and gain material.601
Some interesting electromagnetic effects can only be generated
through well-structured ordering of plasmonic materials, a
property that viruses can provide. A recent example exploring
this potential involved the precise placement of plasmonic
gold nanoparticles with defined spacing and symmetry using
cysteine-functionalized CPMV.281 The CPMV was engineered
to allow gold to be placed at the 12 vertices around the capsid.
The resultant plasmonic nanocluster demonstrated resonances
at visible wavelengths due to interactions of neighboring
Fig. 26 Controlled display of gold nanoparticles and fluorophores for
enhanced fluorescence. (a) Schematic for the assembly of MS2 around
gold nanoparticles followed by attachment of DNA hairpins to place
fluorophores a fixed distance away from the capsid. (b) Images from total
internal reflection fluorescence microscopy of MS2 labeled with fluoro-
phores set 3 bp away from the capsid, with (left) and without (right) gold
encapsulated, demonstrating metal-enhanced fluorescence. Reproduced
with permission from ref. 174. Copyright 2013 American Chemical Society.
This journal is © The Royal Society of Chemistry 2016
 View Article Online

Review Article Chem Soc Rev

gold nanoparticles, and finite-element simulations suggest the
structure is likely to give rise to a 10-fold enhancement of the
local electromagnetic field through near-field coupling.
5. Summary and future directions
Overall, viruses have been exploited in the development of
a dizzying array of applications that fall under the broad scope
of medicine, biotechnology, and energy. While some areas in
medicine, such as vaccines and gene delivery, have been
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and CHE-1306447 to NFS for virus:polymer chemistry, CMMI-
1333651 for virus nanomanufacturing), the National Institutes
of Health (F31 HL129703 to AMW, R03EB020602 to NFS for
shape engineering of VNPs, R01CA202814 and R21EB020946 to
NFS for imaging, R21HL121130 to NFS for cardiovascular
targeting), the Department of Energy (BES DE-SC0008068 to
NSF for basic energy science), the American Cancer Society
(128319-RSG-15-144-01-CDD to NFS for drug delivery), and
the Susan G. Komen Foundation (CCR14298962 to NFS for
vaccines).

around for a while, the application of viruses in other areas,

such as drug delivery and tissue engineering, have only recently
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